Glutathione

ABSTRACT

The use of glutathione to treat or prophylax smallpox infection, to treat or prophylax chemical warfare agents, or to treat or prophylax radiation injury to humans due to release of radioactive elements is proposed. The preferred formulation is an oral dosage form, with reduced glutathione stabilized by ascorbic acid in at least equimolar concentration. The formulation may be in unit dosage form or in bulk.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of priority under 35 U.S.C. §119(e) from U.S. Provisional Patent Application No. 62/182,229, filed Jun. 19, 2015, the entirety of which are each expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of glutathione for treatment and/or prophylaxis of various conditions.

BACKGROUND OF THE INVENTION

The ubiquitous tripeptide L-glutathione (GSH) (gamma-glutamyl-cysteinyl-glycine), is a well known biological antioxidant, and in fact is believed to be the primary intracellular antioxidant for higher organisms. When oxidized, it forms a dimer (GSSG), which may be recycled in organs having glutathione reductase. Glutathione may be transported through membranes by the sodium-dependent glutamate pump. Tanuguchi, N., et al. Eds., Glutathione Centennial, Academic Press, New York (1989), expressly incorporated herein by reference.

Glutathione (L-gamma glutamylcysteinyl glycine, reduced) is widely distributed in Nature, including yeast cells, botanic life and animals. It is made in the same way in humans by two different enzymes, and this is relevant to understanding the properties of glutathione (12).

First, glutamic acid, with carboxylic acid groups on its alpha and its gamma carbons, is held by the first enzyme, gamma glutamyl cysteine synthase, which activates the gamma carboxylic acid to form a gamma amino peptide bond with cysteine, rather than the usual amino peptide bond from a carboxylic acid to a amino of the next amino acid. This gamma amino peptide bond results in a distribution of molecular charges that differs from the usual amino peptide bonds, by creating a zwitterion at the glutamyl terminus, around the alpha carbon.

The second step is the addition of glycine by glutathione synthase, via the usual alpha-to-alpha bond (12). Glycine is critical to prevent the otherwise rapid oxidation of the gamma glutamyl cysteine dipeptide. Evolutionary selection put glycine at this site, rather than a slightly larger amino acid such as alanine, which has a methyl group replacing the hydrogen on the simple, small glycine. The alanine methyl group would cause too much steric hindrance and overly restrict the ability of glutathione to react.

In molecular models, the gamma link provides specific controls over the exposure of the thiol of cysteine, the distribution of molecular charges and determines the intramolecular movements of the flanking amino acids that provide steric hindrance against promiscuous reactions of the thiol. In models with alpha-to-alpha links, the thiol exposure, distribution of molecular charges and intramolecular movements differ from those of the gamma-to alpha link.

It is clear that this molecule is “bent” in the same way across thousands of life forms, by two enzymes, and has evolved over a long time to specifically position its critical thiol to safely support life forms, from simple to complex. It is the configuration of this form of glutathione that makes it so effective at the epicenter of several life-sustaining processes and explains the seven powerful properties of glutathione detailed below. For comparison, the thiol of cysteine, alone, is overexposed and over reactive, and the thiol of homocysteine even more so, creating a highly destructive sulfur radical that disrupts endothelium in homocysteinemia. N-acetyl cysteine (NAC), when taken orally, looses the N-acetyl in the stomach, thereby leading to uncontrolled oxidation and a range of toxicities in humans.

The properties of glutathione (“GSH”) derive from just a few central facts including the molecular configuration of L-gamma glutamylcysteinyl glycine, its controlled reactivity, its ability to maintain a physiologically favorable Redox potential, its antioxidant properties in all subcellular compartments, the existence of avid glutathione transporters on cell membranes and mitochondria and the fact that these properties are supported by enzymes: (i) those that synthesize GSH; (ii) enzymes that amplify particular properties, such as GSH peroxidases and S-transferases and; (iii) enzymes that restore GSH after it has been used, GSH reductase. The properties of glutathione have been categorized into four groups, but can be organized differently.

GSH is known to function directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells from free-radical mediated damage. GSH is one of the primary cellular antioxidants responsible for maintaining the proper oxidation state within the body. GSH is synthesized by most cells, and is also supplied in the diet. GSH has been shown to recycle oxidized biomolecules back to their active, reduced forms.

Because of the existing mechanisms for controlling interconversion of reduced and oxidized glutathione, an alteration of the level of reduced glutathione (GSH), e.g., by administration of GSH to an organism will tend to shift the cells of the organism to a more reduced redox potential. Likewise, subjecting the organism to oxidative stress or free radicals will tend to shift the cells to a more oxidized potential. It is well known that certain cellular processes are responsive to redox potential.

Reduced glutathione (GSH) is, in the human adult, produced from oxidized glutathione (GSSG) primarily by the liver, and to a smaller extent, by the skeletal muscle, red blood cells, and white cells. About 80% of the 8-10 grams glutathione produced daily is produced by the liver and distributed through the blood stream to the other tissues.

A deficiency of glutathione in cells may lead to excess free radicals, which cause macromolecular breakdown, lipid peroxidation, buildup of toxins, and ultimately cell death. Because of the importance of glutathione in preventing this cellular oxidation, glutathione is continuously supplied to the tissues. However, under certain conditions, the normal, physiologic supplies of glutathione are insufficient, distribution inadequate or local oxidative demands too high to prevent cellular oxidation. Under certain conditions, the production of and demand for glutathione are mismatched, leading to insufficient levels on an organismal level. In other cases, certain tissues or biological processes consume glutathione so that the intracellular levels are suppressed. In either case, by increasing the serum levels of glutathione, increased amounts may be directed into the cells. In facilitated transport systems for cellular uptake, the concentration gradient which drives uptake is increased.

As with all nutrients, eating or orally ingesting the nutrient would generally be considered a desired method for increase body levels thereof. Thus, attempts at oral glutathione treatments were known, and indeed the present inventors hereof previously suggested oral glutathione administration for various indications. The protocols for administration of glutathione, however, were not optimized and therefore the bioavailability of the glutathione was unassured and variable. Prior pharmaceutical attempts by others to safely, effectively and predictably raise intracellular GSH through oral therapy with GSH have not met with demonstrated success. Experts generally believe that beneficial physiological effects of orally administered glutathione are difficult or impossible to achieve, or the efficiency is so low as to make supplementation by this route unproductive.

Because of the poor or variable results obtained, the art generally teaches that oral administration of glutathione is ineffective, forcing administration or supplementation by other routes, principally intravenously, but also by alveolar inhalation. Orally absorbed prodrugs and precursors have also been proposed or used. A known pharmacological regimen provides intravenous glutathione in combination with another agent, such as cis-platinum (a free radical associated metal drug), doxorubicin, or daunorubicin (free radical associated drugs which interact with nucleic acid metabolism), which produced toxic side effects related to free radical reactions.

The ability to harness GSH, which is a powerful, but safe substance, into an effective oral pharmaceutical had not been accomplished in the past, because of molecular instability, poor gastrointestinal absorption through existing protocols and resulting inability to reliably affect increases in intracellular GSH levels. Administering sufficient amounts to achieve physiological benefit using known oral administration protocols might lead to cysteine related kidney stones, gastric distress or flatulence.

Glutathione is relatively unstable in alkaline or oxidative environments, and is not absorbed by the stomach. It is believed that glutathione is absorbed, after oral administration, if at all, in the latter half of the duodenum and the beginning of the jejunum. It was also believed that orally administered glutathione would tend to be degraded in the stomach, and that it is particularly degraded under alkaline conditions by desulfurases and peptidases present in the duodenum. Thus, known protocols for oral administration of glutathione involved administered with meals or after eating to buffer pH extremes and dilute degradative enzymes. This protocol, however, has the effect of diluting the glutathione and delaying absorption. Studies directed at determining the oral bioavailability of glutathione under such circumstances showed poor absorption, and therefore such administration was seen as of little benefit.

Therefore, while oral dosage forms of glutathione were known, the clinical benefits of these formulations were unproved and, given the lack of predictability of their effect, these formulations were not used for the treatment of specific conditions, nor proven to have effect. Further, the known protocols for administration of glutathione did not provide convenience and high bioavailability.

The prior art thus suggests that glutathione esters might be suitable as orally bioavailable sources of glutathione, which are stable and may be rapidly absorbed. However, these are both more expensive than glutathione itself and have proven toxic.

Pure glutathione forms a flaky powder that retains a static electrical charge, due to triboelectric effects, making processing and formulation difficult. The powder particles may also have an electrostatic polarization, which is akin to an electret. Glutathione is a strong reducing agent, so that autooxidation occurs in the presence of oxygen or other oxidizing agents. U.S. Pat. No. 5,204,114, Demopoulos et al., expressly incorporated herein by reference in its entirety, provides a method of manufacturing glutathione tablets and capsules by the use of crystalline ascorbic acid as an additive to reduce triboelectric effects which interfere with high speed equipment and maintaining glutathione in a reduced state. A certain crystalline ascorbic acid is, in turn, disclosed in U.S. Pat. No. 4,454,125, Demopoulos, expressly incorporated by reference herein in its entirety. This crystalline form is useful as a lubricating agent for machinery. Ascorbic acid has the advantage that it is well tolerated, antioxidant, and reduces the net static charge on the glutathione.

In synthesizing glutathione in the body, cysteine, a thiol amino acid is required. Since the prior art suggests that oral administration of glutathione itself would be ineffective, prodrugs or precursor therapy was advocated. Therefore, the prior art suggests administration of cysteine, or a more bioavailable precursor of cysteine, N-acetyl cysteine (NAC). While cysteine and NAC are both, themselves, antioxidants, their presence competes with glutathione for resources in certain reducing (GSH recycling) pathways. Since glutathione is a specific substrate for many reducing pathways, the loading of a host with cysteine or NAC may result in less efficient utilization or recycling of glutathione. Thus, cysteine and NAC are not ideal GSH prodrugs. NAC has also demonstrated some neurotoxicity. Thus, while GSH may be degraded, transported as amino acids, and resynthesized in the cell, there may also be circumstances where GSH is transported into cells without degradation; and in fact the administration of cysteine or cysteine precursors may interfere with this process.

A number of disease states have been specifically associated with reductions in glutathione levels. Depressed glutathione levels, either locally in particular organs, or systemically, have been associated with a number of clinically defined diseases and disease states. These include HIV/AIDS, diabetes and macular degeneration, all of which progress because of excessive free radical reactions and insufficient GSH. Other chronic conditions may also be associated with GSH deficiency, including heart failure and coronary artery restenosis post angioplasty.

Clinical and pre-clinical studies have demonstrated the linkage between a range of free radical disorders and insufficient GSH levels. Newly published data implies that diabetic complications are the result of hyperglycemic episodes that promote glycation of cellular enzymes and thereby inactivate GSH synthetic pathways. The result is GSH deficiency in diabetics, which may explain the prevalence of cataracts, hypertension, occlusive atherosclerosis, and susceptibility to infections in these patients.

GSH functions as a detoxicant by forming GSH S-conjugates with carcinogenic electrophiles, preventing reaction with DNA, and chelation complexes with heavy metals such as nickel, lead, cadmium, mercury, vanadium, and manganese. GSH also plays a role in metabolism of various drugs, such as opiates. It has been used as an adjunct therapy to treatment with nephrotoxic chemotherapeutic agents such as cisplatin, and has been reported to prevent doxorubicin-induced cardiomyopathy. GSH is also an important factor in the detoxification of acetaminophen and ethanol, two powerful hepatotoxins. See: Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on the decomposition of glutathione. I. Decomposition in solid state. Chem. Pharm. Bull. 26:2081-91, 1978.; Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition of glutathione. II. Anaerobic decomposition in aqueous solution. Chem. Pharm. Bull. 28:514-20, 1980.; Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition of glutathione. III. Peptide bond cleavage and desulphurization in aqueous solution. Chem. Pharm. Bull. 28:521-28, 1980.; Hagen, T. M., Aw, T. Y., and Jones, D. P.: Glutathione uptake and protection against oxidative injury in isolated kidney cells. Kidney Intl. 34:74-81, 1988.; Lash. L. H., and Jones, D. P.: Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma. Arch. Biochem. Biophys. 240:583-92, 1985.; Meister, A.: Selective modification of glutathione metabolism. Science 220:472-477, 1983.; Meister, A. and Anderson, M. E.: Glutathione. Ann. Rev. Biochem. 52:711-60, 1983.; Riley, R. J., Spielberg, S. P., Leeder, J. S.: A comparative study of the toxicity of chemically reactive xenobiotics towards adherent cell cultures: selective attenuation of menadione toxicity by buthionine sulphoximine pretreatment. J. Pharmacol. 45(4): 263-267, 1993.; Wierzbicka, G. T., Hagen, T. M. & Jones, D. P.: Glutathione in food. J. Food Comp. Anal. 2:327-337, 1989.; Bravenboer, B., Kappelle, A. C., Hamers, F. P., van Buren, T., Erkelens, D. W. & Gispen, W. H.: Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozocin-induced diabetic rat. Diabetologia 35:813-817, 1992.; Cavaletti, E., Tofanetti, O. & Zunino F.: Comparison of reduced glutathione with 2-mercaptoethane sulfonate to prevent cyclophosphamide-induced urotoxicity. Cancer Letters 32:1, 1986.; Hamers, F. P., Brakkee, J. H., Cavalletti, E., Tedeschi, M., Marmonti, L., Pezzoni, G., Neijt, J. P. & Gispen, W. H.: Reduced glutathione protects against cisplatin-induced neurotoxicity in rats. Cancer Res. 53:544-549, 1993.; Kromidas, L., Trombetta, L. D., and Jamall, I. S.: The protective effects of glutathione against methylmercury cytotoxicity. Toxicol. Letters 51:67-80, 1990.; Novi, A. M., Flohe, R., and Stukenkemper, S.: Glutathione and aflatoxin B₁-induced liver tumors: requirement for an intact glutathione molecule for regression of malignancy in neoplastic tissue. Ann. NY Acad. Sci. 397:62-71, 1982.; Rao, R. D. N., Fischer, V., and Mason, R. P.: Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. J. Biol. Chem. 265:844-7, 1990.; Skoulis, N. P., James, R. C., Harbison, R. D. and Roberts, S. M.: Depression of hepatic glutathione by opioid analgesic drugs in mice. Toxicol. Appl. Pharmacol. 99:139-47, 1989.; Villani, F., Galimberti, M., Zunino, F., Monti, E., Rozza, A., Favalli, L. & Poggi, P.: Prevention of doxorubicin-induced cardiomyopathy by reduced glutathione. Cancer Chemother. Pharmacol. 28:365-369, 1991.; Wagner, G., Frenzel, H., Wefers, H. and Sies, H.: Lack of effect of long-term glutathione administration on aflatoxin B1-induced hepatoma in male rats. Chem. Biol. Interactions 53:57-68, 1985.; Yoda, Y., Nakazawa, M., Abe, T. & Kawakami, Z.: Prevention of Doxorubicin myocardial toxicity in mice by reduced glutathione. Cancer Research 46:2551, 1986.; Younes, M., and Strubelt, O.: Protection by exogenous glutathione against hypoxic and cyanide-induced damage to isolated perfused rat livers. Toxicol. Letters 50:229-236, 1990.; McCartney, M. A.: Effect of glutathione depletion on morphine toxicity in mice. Biochem. Pharmacol. 38:207-9, 1989.; Ishida, T., Kumagai, Y., Ikeda, Y., Ito, K., Yano, M., Toki, S., Mihashi, K., Fujioka, T., Iwase, Y. and Hachiyama, S.: (8S)-(glutathion-S-YL)dihydromorphinone, a novel metabolite of morphine from guinea pig bile. Drug. Metab. Dispos. 17:77-81, 1989.; Nagamatsu, K., Kido, Y., Teroa, T, Ishida, T. and Toki, S.: Protective effect of sulfhydryl compounds on acute toxicity of morphinone. Life Sci. 30:1121-27, 1982.

SUMMARY OF THE INVENTION

Pharmaceutical glutathione (GSH) is safe, stable, rapidly bioavailable and replenishes GSH lost continuously in viral infections, exposures to toxic chemicals, and radiation. It maintains intracellular levels needed to express four properties that are concentration dependant. GSH therapy: (1) assures glutathione availability to support TH1 immunologic responses needed to recover from smallpox; (2) slows activation and over-expression of NF-κB and inflammatory cascades that cause cumulative tissue toxicities; (3) biochemically neutralizes reactive intermediates that otherwise cause cellular and tissue toxicities; (4) may disable the activities of key viral proteins, and curb replication. As a result, GSH therapy may decrease morbidity and possibly mortality, as shown in examples of human and animal studies of HIV infection, other pox viruses, and hepatitis C. Each of these is described in more detail below.

Consistently normal intracellular concentrations of GSH help maintain the balance of T Helper 1 and 2 (TH1 and TH2) immunologic response patterns. When GSH is continuously lost, restorative GSH therapy rapidly up-regulates TH1, enhancing Interferon γ and cell mediated immunity required for recovery, while down-regulating IL-4, a disadvantageous TH2 cytokine, when over expressed during acute viral infections. This beneficial effect of GSH, required for recovery responses, has been demonstrated against other dangerous viruses, including pox viruses.

Consistently normal intracellular concentrations of glutathione also sets a high reduction oxidation potential within cells, that slows activation and over-expression of NF-κB, TNFα, IL-1β, adhesion molecules, cyclo-oxygenase-2, matrix metalloproteinases and inflammatory cascades. This mechanism has also been demonstrated against other dangerous viruses, including pox viruses. The ability to help control such reactions indicates further, potential uses of GSH in counter terrorism.

Biochemical neutralizing reactions: (a) GSH neutralizes reactive oxygen and reactive nitrogen species (ROS and RNS) continuously produced during viral infections that otherwise damage cell membrane lipids, proteins, and nucleic acids, and result in cellular and tissue toxicities; (b) GSH protects mitochondria against hydrogen peroxide, bioenergetic failure, and exaggerated, apoptotic processes that add to cumulative tissue toxicities; (c) Consistently normal intracellular concentrations of GSH help control non enzymatic and enzymatic oxidations of arachidonic acid. Otherwise these cause tissue-disrupting excesses of reactive intermediates from lipid hydroperoxides (alkoxy radicals, LO., and hydroxyls, .OH). The ability to help control such reactions indicates further potential uses of GSH in counter terrorism.

(4) As occurs with GSH when used against HIV, the thiol (—SH) moiety is capable of post translational protein modifications that may disable thermodynamically less stable viral proteins synthesized during infections. For example, variola proteins that may be hindered include those involved in viral replication, for example, encoded viral DNA topoisomerases, and those involved in evasion of host immunologic defenses.

SUMMARY OF BASIC CLINICAL PHARMACOLOGY OF GLUTATHIONE

Smallpox is categorized a Class A Bioterror Agent along with Bacillus anthracis, Yersinia pestis, Francisella tularensis, Botulinum toxin, and the Filo and arenaviruses such as Ebola and the Hemorrhagic fevers. Experts believe that only a few virions are necessary for transmission of smallpox (3). Somewhat of an uncertainty is the thought of bioengineered smallpox with additional gene copies of Interleukin-4 (IL-4), a cytokine, which, if over-expressed, suppresses IL-12, INF γ, and specific cell mediated immunity, the critical factors needed for survival and recovery from an acute viral infection. The landmark efforts of Henderson and colleagues (3) eradicated natural smallpox infections through very hard work and “ring vaccinations”. However, the covert dissemination of weaponized smallpox remains a threat.

Pharmaceuticals that serve to decrease morbidity and mortality would help to supplement the successful management of smallpox bioterrorism, biowarfare and its threats. Safe drugs “fill in” where there may be logistical encumbrances to orderly, “screened” vaccinations. In the overall strategy vs. smallpox terrorism, an important point is to deliver and administer vaccine to the possibly exposed individuals within the “seven day window” of incubation. Pharmaceutical glutathione is safe and can “fill in” where there is a doubt as to whether an individual is within the seven-day period after initial exposure. Some of the individuals in Nursing Homes are elderly, incapacitated and towards the end of life. They may be likened to the people in the past global public health initiative vs. smallpox who were severely undernourished and described as having “significantly low serum albumin . . . protein depletion . . . low peripheral blood lymphocyte count and spectacular unresponsiveness to many antigens (8). Individuals in these circumstances most likely will not receive the smallpox vaccine since efficacy will be diminished, and the complication rates will be higher. The use of pharmaceutical glutathione in these circumstances may prove helpful. Pharmaceutical glutathione can serve as part of a comprehensive shield to augment education, and vaccination. This addition is safe, scientific and rapidly implementable.

The administration of pharmaceutical glutathione would not hinder the efficacy of vaccination, rather it would help in view of the favorable effects of glutathione in fostering Th1 over Th2 immunologic response patterns described and referenced below. Ennis et al in 2002 (9) found that smallpox vaccine given by bifurcated needle induces strong vaccinia virus-specific CD8 (+) CTL and IFN-gamma-producing T cell responses (9). These are the responses that glutathione enhances, provided it is present in normal intracellular concentrations within monocytes/macrophages, dendritic cells and B lymphocytes. The preferred formulation of GSH, for example as set forth in U.S. Pat. No. 5,204,114, expressly incorporated by reference, or bulk packed without unit dosage formulation, is well absorbed, and distributed into the Peripheral Blood Mononuclear Cells (PBMC's) starting within 0.5 hours after oral ingestion.

Central to the pharmaceutical-based reduction of morbidity for people beyond the “seven day window” of the vaccine and others within “the herd”, is the question of the major processes comprising the molecular pathology of smallpox at the tissue and cellular levels, host factors responsible for progression, and cause(s) of death. Francis in 2002 (10) published his findings on the cause of death in smallpox. He reviewed the surviving case series of smallpox pathology in humans as well as other review articles from English language journals written during the last 200 years . . . . The cytopathic effects of smallpox cause death. The data did not support previously promulgated theories attributing death to a bacterial sepsis syndrome seeded from the pustules . . . in a future outbreak, antibiotic therapy would minimally influence mortality (10). If cases of smallpox develop, the enhanced supportive care available now will likely decrease morbidity and mortality. Beyond that there is no established proactive therapy for smallpox infections. High doses of pharmaceutical glutathione would serve in a safe, proactive capacity, as the discussion of the four properties of glutathione, and the examples of several other serious viral infection detailed below, will show. The drug should be able to replenish and maintain intracellular glutathione concentrations and thereby interdict smallpox pathology at the tissue, cellular, and molecular levels. The dosing is oral and safe.

The review by Henderson and the Working Group on Civilian Biodefense (3) provides the same view as Francis (10) with regard to secondary bacterial infection. In the Henderson review (3), the following is noteworthy: Secondary bacterial infection is not common, and death, which usually occurs during the second week of illness, (this is approximately 3-4 weeks after initial exposure) most likely results from the toxemia associated with circulating immune complexes and soluble variola antigens The CDC, in a medical education satellite broadcast and videotape (11) points out: physiologic host factors make the difference in a case and how severe it will be; slow progression of the rash . . . compared to other rash illnesses is a smallpox characteristic; the infection is now known to involve multiple organs; overwhelming viremia; and toxemia contribute to death.

Pharmaceutical glutathione, based on: the literature; its four properties; its effects against other dangerous viruses; and, the specific formulation that makes glutathione chemically stable, safe, and rapidly bioavailable so as to replenish and maintain intracellular glutathione concentrations, should be able to do the following: enhance defensive, physiologic host immunologic reactions, specifically Th1 response patterns; exercise its properties effectively and quickly with simple oral administration, since the disease progresses slowly with an approximate two week period from exposure to rash, with subsequent slow development of the rash; in this time period, glutathione should be able to interdict full development of the infection, perhaps converting it to a mild form comparable to smallpox occurring in a person vaccinated (10 years-20 years earlier, or to alastrim, i.e., variola minor (3)); distribute systemically following oral administration, since glutathione transporters are present on cell surfaces; glutathione transporters are on cell membranes as well as in mitochondria, and are discussed and referenced in further sections; increase IFN γ, which in turn induces nitric oxide synthase (iNOS), which increases the potent antimicrobial .NO (nitric oxide) that clears viremias; reduce toxemia related to excessive, pro inflammatory cytokines since glutathione can suppress NFκB activation, along with TNFα, interleukin 1 b, COX-2 and other cytokines contributing to the “toxic” state of the patient.

The sequential clinical presentation of smallpox, (2, 3), starting from exposure, to asymptomatic viral replication with two early viremias, to prodroma, to rash, and then to complications, or recovery, or demise reveals unique stepwise pathogenesis: blocked initial host defense cellular reactions accompanied by unimpeded and asymptomatic viral replication with viremias, and later on, cascades of severe unregulated inflammatory reactions, localization of adherent variola-laden leucocytes within dermal capillaries, leucocyte diapedesis from the capillaries to infect the skin, destructive rash, toxemias, circulating immune complexes with soluble variola antigens, and complement activation, among others.

Following is the sequential, clinical outline of the pathogenesis described by Henderson and colleagues (3), and by others (2), accompanied by a parallel, stepwise delineation of specific molecular responses that are blocked, inadequate, or unregulated, but safely correctible, in part, by high dose pharmaceutical glutathione.

Poxviruses, including variola, normally encode proteins for replication, such as DNA topoisomerase (149-154), and others, which when expressed by the infected host cells, blunt host immunologic strategies to contain and eradicate the infection, including blocking NFκB activation, chemokines, cytokines, Interferon gamma (IFNγ), complement fragments, and cell mediated immunity (155-165). Further, weaponized (1), biologically engineered pox variants can be instilled with added, encoded proteins that may blunt the immunologic strategies of immunized hosts (14, 24, 25), and lead to patterns of disease spread and severity not previously encountered.

Administration of glutathione (GSH) offers protective mechanisms through its properties, detailed and referenced previously, that can safely counter the variola pathology in this phase. GSH is known to up-regulate T helper 1 (Th1) response patterns and may, if used early, increase Interleukin-12 (IL-12), IL,-18, IFNγ, enhance specific cytotoxic lymphocytes (13, 15, 16, 18-23), enhance NK cell activity (44), and increase Redox-sensitive protective responses (15, 16, 44, 46-51, 54-63).

In the early stage of asymptomatic variola infection, many of the dendritic cells, macrophages and lymphocytes have not yet become infected and remain optimally responsive to GSH; however, host responses may be blocked by expressed viral proteins that are soluble and secreted. These can adversely affect neighboring cells that are as yet uninfected. For example, Alcami, Symons and G. L. Smith, in 2000 (166), presented novel actions of some of the poxvirus evasion proteins they had studied in their laboratories at the Sir William Dunn School of Pathology. They noted Poxviruses encode . . . soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-γ), IFN-α/β, and chemokines. These . . . have a profound effect on virus pathogenesis (166). These soluble viral proteins were found to bind to both uninfected and infected cells, thereby nullifying the protective, anti-viral effects of interferons and other host reactions . . . the soluble viral proteins serve as receptors as they bind to all cell surfaces, assuring unimpeded viral access and entry to new, uninfected cells (166). The infection therefore neutralizes host cell defenses even before the new cells have become infected. The counter strategy, in addition to timely quarantines, education and vaccinations can now include GSH as a safe pharmaceutical that: (a) raises numerically challenging quantities of defensive biomolecules vs. the viral proteins by up-regulating Th1 responses, (b) increases Redox-sensitive defense cells, like NK cells, and (c) attacks the sensitive conformational disulfide bonds of viral evasion and replicatory proteins.

The fact that the smallpox vaccine is highly effective from the acute administration within seven days of likely exposure, among those that can safely take the vaccine and are assuredly within the 7 days, means that uninfected, or perhaps even the early-infected cell populations, can rapidly produce sufficient quantities of IL-12, IL-18, IFN gamma, and factors to stimulate NK cells, and variola-specific CTL's to overcome the viral evasion proteins and STOP the infection (2, 3, 9, 11) in this early phase. Ennis et al in 2002 (9) found that smallpox vaccine given by bifurcated needle induces strong vaccinia virus-specific CD8+ CTL, and IFN-gamma-producing T cell responses (9). These are the same biochemical and cell responses that glutathione rapidly enhances by fostering Th1 responses (13, 15, 16, 18-23), NK cell activity (44), and protective Redox-sensitive responses (15, 16, 44, 46-51, 54-63).

The pox proteins react directly and biochemically with host proteins, often via thiols and disulfides. Protein-protein interactions occur normally, and in other pathologic states. Some tend to be stochiometric, and others not. This provides a rationale for the early use of high dose glutathione treatment to quantitatively enhance host immune responses and numerically overwhelm the blocking pox virus protein(s). Upregulation of Th1 response patterns includes development of specific cytotoxic lymphocytes (CTL's) to kill and clear variola infected cells.

Natural infection occurs following implantation of the virus on the oropharyngeal or respiratory mucosa (3). The infectious dose is unknown but is believed to be only a few virions. After the migration of the virus to and multiplication in regional lymph nodes, an asymptomatic viremia develops on about the third or fourth day, followed by multiplication of virus in the spleen, bone marrow, and lymph nodes (3).

This helps to eliminate the source of the offending pox proteins and represents an advantageous, non stochiometric result in favor of the host. GSH is non toxic, as manufactured by ThyoGen and Kyowa Hakko, Co., Ltd. In ThyoGen's Phase 1/2 studies, an increasing range of oral doses provided safe, significant, dose related increases in GSH in the Peripheral Blood Mononuclear Cells (PBMC's) (see Sections 7, 8 and 9).

Another useful property of GSH, in addition to fostering Th1 pathways and Redox-sensitive protection, involves GSH and direct post translational modifications of viral proteins. GSH, as a function of changes in concentrations and in Redox Potentials, regulates proteins, cell signaling and other reactions by reducing disulfide bonds, or by glutathionylating the thiols of cysteine residues. This unfolds many proteins, and generally, inhibits enzymes and ligand binding to receptors. Unfolding of proteins may also expose crucial tyrosine sites that may be advantageously nitrosylated into Nitrotyrosine, for example, within an activated macrophage producing nitric oxide that can now attack the active site tyrosine in viral DNA topoisomerase (140-144, 149-154).

The probability of decreased thermodynamic stability of viral proteins expressed by the infected host cells, compared to host proteins, (140-154), provides an interventional opportunity, based on the lower stability. Examples of this property, specifically in viral infections, includes, among other HIV examples, glutathionylating HIV protease (148). Two conserved cysteines of HIV-1 protease (Cys-67, Cys-95) are readily glutathionylated, resulting in an enzyme preparation with significantly lower specific activity (148). Removal of the glutathione restored the full activity of the enzyme preparations. Examples of inactivating disulfide bonds are studies of HIV gp120 (146). Replicating HIV-1 with glutathione exposure, in vitro, resulted in the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds (147). The effect of GSH on HIV gp120, was previously demonstrated in studies showing decreased viral infectivity, in vitro (146). The authors related this to selective inhibition of envelope glycoproteins, specifically the major such protein, gp120, rich in intra chain disulfide bonds These GSH modifications of viral proteins are achieved without cytotoxicity (145-148).

The “secondary” viremia, early symptomatic phase, with fever and toxemia, occurs at days 8-14 post exposure, and herald the activation and loss of control of the NFκB family of transcription factors. The oxidative stress accompanying infections, the NADPH activation on macrophage plasma membranes, and the GSH consumption activate histone acetylation (48), and DNA unwinding that gives access to the extensive gene network that is NFκB dependent (54).

Meanwhile, NFκB has been activated (15, 16, 47, 54, 55) and there is excess production of: proinflammatory cytokines including Th1 cytokines IL-12 and IFNγ (13, 15, 16, 18-23), IFNγ-dependent factors (34-43), IL-18 (56), and Th2 cytokines IL-4 (14, 24, 25); excess production of IL-4 (14, 24, 25); excess production of adhesion molecules (57-60); induction of matrix metalloproteinases (61-63); uncontrolled cyclooxygenase-2 (COX-2) (47); accelerated synthesis of eicosanoids (82); activation of inducible nitric oxide synthase (iNOS) (34-36); chemokines (50,51), among other factors in the gene network of NFκB (54). These reactions, such as uncontrolled lipid peroxides (LOOH's) (73-75, 81, 82, 93-95, 136-139); excesses of RNS (14, 98-103); and the free radical reactions engendered by TNFα (15, 16, 83-88) erode glutathione concentrations. This leads to further declines in the Redox potential, and further exaggeration of the acute inflammatory reactions. Th1 response patterns decline further, and IL-12 INFγ are suppressed as GSH levels fall. The non-development of specific cytotoxic lymphocytes (CTL's) is part of the down-regulated Th1 response patterns and results in the unimpeded spread of variola-infected mononuclear cells.

The expression of adhesion molecules on the infected mononuclear cells and on activated endothelium causes adherence of the mononuclear cells to injured endothelium. This is followed by diapedesis of the infected cells through the injured capillaries to form perivascular infiltrates in the dermis. Variola viruses then emerge to infect and destroy the skin cells, particularly those of the sebaceous glands of the face and “the bathing suit” regions (2,3). SEM images by H. B. Demopoulos and colleagues (69,70), who utilized a model of regional cerebral ischemia to initiate free radical mediated damage and to cause “activation” of endothelium, with apparent production of adhesion molecules and leucocyte-vessel wall interactions, are surrogate examples of the type of leucocyte-vessel wall interactions that might be seen with variola-infected mononuclear cells adhering and eventually penetrating into the tissues. There is a reasonable surrogate example of pathologic processes that involve NFκB activation, its cascades, including adhesion molecules such as ICAM-1. In 2002, Berti and colleagues at the Walter Reed Army Institute of Research (167) performed middle cerebral artery occlusions (MCAO) in Sprague-Dawley rats and measured inflammatory gene expression, including adhesion molecules, in cerebral ischemia, using quantitative real-time RT-PCR analysis (167). They noted Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules cytokines, some of which are regulated by . . . NFκB . . . the induced molecules, which are involved in the initiation of the inflammatory cascade . . . may thus contribute to secondary cellular responses that lead to further brain damage (167).

Various types of pathologic changes occur in blood vessels that are damaged by oxidative stress (lipid peroxides, reactive oxygen species, reactive nitrogen species), as occurs in serious infections, ischemia, and other conditions characterized by glutathione depletion.

The junction between two endothelial cells is prominently seen in SEM. It is a “Tight Pavement” so nothing can leak. It is a slick, non-stick pavement so that blood flows smoothly. These lining cells actively produce molecules (e.g., Nitrous Oxide and PGI₂) that keep the blood vessels wide open and non stick. Glutathione is mandatory to help Nitrous Oxide and PGI₂ carry out their critical functions. Otherwise, blood vessels narrow greatly, and blood components, like platelets and white blood cells, “gum up” the vessels. Endothelial blebs and small pores are “normal” in these SEM preparations.

Adherent leucocytes in a small artery, approximate diameter 200 microns, also release substances that injure blood vessels, and some can move through the wall by diapedesis to attack surrounding tissue cells. Occurs in serious infections, ischemia and in other disorders due to elaboration of adhesion molecules on the leucocytes and on the endothelial cells, secondary to decreased GSH. This is the type of process seen when variola laden mononuclear cells lodge in the dermal capillaries preparatory to invading the skin cells.

Some leucocytes seen under SEM have activated their surface NADPH oxidases which produce a variety of directly destructive reactive oxygen species. The effect could create large craters, and other ultra-structural irregularities in the endothelium.

The aftermath of an adherent leucocyte that has caused free radical damage to the endothelium of a small artery can also be seen by SEM. Cratering and the over riding of lining cells can be observed. This occurs in serious infections, ischemia and other disorders characterized by glutathione deficiency. Craters like these are seen only at times when adherent leucocytes are present and may represent direct erosion by ROS and RNS produced by monocytes/macrophages. The over riding of the endothelial cell is probably caused by increased production of matrix metallopreoteinases that affect basement membrane materials and other supportive molecules of blood vessels (61-63).

A secondary viremia begins on about the eighth day and is followed by fever and toxemia. The virus, contained in leucocytes, then localizes in small blood vessels of the dermis and beneath the oral and pharyngeal mucosa and subsequently infects adjacent cells (3). From a series of older sources The typical skin lesion starts with changes in the capillaries of the corium and is characterized by dilation, endothelial proliferation and perivascular mononuclear infiltration. In the adjacent epidermis, reticular degeneration of the cells . . . occurs. The cells swell and the characteristic Guarneri bodies make their appearance. These are spherical bodies lying close to the nucleus . . . (and) . . . consist of collections of virus elementary bodies and (the spherical bodies) range in size from 2 to 8 microns . . . . The swollen cells rupture, forming a vesicle . . . the cells beneath the vesicle undergo a different type of degeneration resembling the “ballooning degeneration” that occurs in chickenpox (2).

Matrix metalloproteinases (MMP's) (collagenases, gelatinases, and others constitute a family of 21 enzymes that attack the known components of the extra cellular matrix) are up-regulated by inflammatory cytokines (61-63) and are operative in the spread of malignancies. The diapedesis of variola-infected mononuclear cells, out of dermal capillaries, and their subsequent spread through the tight collagenous matrix of the dermis, to the epidermal layers, or penetrating through, into sebaceous glandular structures (2,3), are steps that are enhanced by MMP's (61-63). Maintaining high GSH concentrations can help to stem MMP activities and be of material benefit in curtailing the particularly destructive rash.

The cumulative tissue toxicities progress unabated in the approximately 30% to 60% of vaccination-naïve patients who succumb. The survivors make a recovery through a stormy course, and some are left with pitted scars in the skin (2,3). Along with the out-of-balance Th2 response patterns and unregulated inflammatory cascades, that predominate as GSH levels fall, additional pathologic mechanisms are amplified. Reactive Nitrogen Species (RNS), stemming from excessive production of nitric oxide (.NO), combine with Reactive Oxygen Species (ROS) and lipid peroxides (LOOH) to form RNS such as peroxynitrite (.NOO). Lipid peroxidation expands as IL-4 enhances 12- and 15-lipoxygenases, while simultaneously suppressing GSH peroxidases and other enzymes that normally dismantle LOOH's. Host proteins are attacked by the uncontrolled ROS, RNS, alkoxy radicals (LO.) and other reactive intermediates. There is therefore a substantial increase in oxidative stress which erodes glutathione and other antioxidant defenses. Defenses give way as Th2 responses, IL-4, exaggerated inflammatory reactions, and oxidative stress cyclically amplify one another.

The intracellular glutathione concentrations in this phase are probably quite low. There are several inducers of iNOS, including the proinflammatory cytokines, TNFα, IL-1β, and IFNγ. The latter, IFNγ, is probably not a significant factor since there are powerful Th2 processes in effect that serve to suppress IFNγ. In other inflammatory diseases, increased production of IFNγ can have undesirable consequences, since it is a potent activator of iNOS.

By the time smallpox infections reach this imminently life-threatening stage, the number of unregulated and interacting pathways has become very complex. The processes that precede this complex late phase of smallpox infection would be more responsive to restoration of control mechanisms if GSH therapy starts early. However, strong public health measures, education of health professionals and other logistical systems must be in place to identify those who may have been exposed so that screened vaccinations, quarantines, and early glutathione therapy can be instituted.

“Toxic” Phase Day 14 to Day 30 Post Exposure At the one to two week incubation period, (range, 7-17 days), the patient typically experiences high fever, malaise, and prostration with headache and backache (3). A maculopapular rash then appears on the mucosa of the mouth and pharynx, face, and forearms, and spreads to the trunk and legs . . . death, which usually occurs during the second week of illness (approximately the 3^(rd) to 4^(th) week after exposure), most likely results from toxemia associated with circulating immune complexes and soluble variola antigens (3). There is little information about how individuals with different types of immune deficiency responded to natural smallpox infection. Smallpox was eradicated . . . before suitable techniques became available for measuring cell-mediated immunity. However, it is possible that the underlying cause of some cases of malignant and hemorrhagic smallpox resulted from defective immune responses” (3).

The stoichiometry of using GSH, when starting in the late phase, is daunting because of the sheer number of pathways involved. While GSH has controlling properties, it is dependent upon its amplifying enzymes, such as GSH peroxidases, S-tranferases, and GSH reductases. These enzymes are subject to the same oxidative, free radical damage as are other proteins, with the formation of carbonyls. Mitochondrial oxidative phosphorylation is also at risk when GSH levels decline, and lead to impaired synthesis of cellular macromolecules and to apoptosis. Apoptotic losses of macrophages, and antigen processing cells may be avoidable when low mitochondrial GSH levels are responsible. In vitro, GSH additions can prevent threatening mitochondrial induced apoptosis.

In overall summary, the maze of complex interactions described above have a central feature. They run out of control, and this is due, to some significant extent, to the heightened demands on a molecule that has limited availability to begin with . . . glutathione. It is normally in high demand in all tissues, but past 45 years of age even apparently healthy individuals become comparatively deficient. GSH insufficiencies are worsened by tobacco, alcohol, intercurrent infections, diabetes mellitus, the use of pharmaceuticals such as the anti inflammatory corticosteroids, and being over 45 years of age.

The inventor has succeeded in producing a safe, stable, orally bioavailable pharmaceutical glutathione. With its platform of four powerful properties, it has the ability to maintain and to help restore order over some of the dangerous, cumulative tissue toxicities associated with smallpox infections if started early. Shattered biomolecules, apoptogenic cell fragments, toxic by-products of lipid peroxidation, collapse of mitochondrial bioenergetics, and other progressive, irreparable, late pathologic events must be averted. Glutathione will help and will fit well with classic, proven public health measures of education, containment, vaccinations, enforcement of quarantines, and identifications of those in need of glutathione, in the unfathomable inhumanity of The deliberate reintroduction of smallpox as an epidemic disease (which) would be an international crime of unprecedented proportions, but it is now regarded as a possibility (3).

The Four Properties of Glutathione

The Four Properties Comprise the Major Clinical Pharmacology of Glutathione As It Relates to Decreasing the Morbidity, and Possibly, the Mortality of Smallpox Infections. The Pharmacology Broadens in Other Situations. The Categorization Into Four Properties Has Indistinct Boundaries Because They Are Strongly Inter-related.

1. High normal GSH concentrations in dendritic cells, macrophages and lymphocytes rapidly up-regulate T helper 1 (Th1) response patterns (ex. IL-12, Interferon gamma, and specific cell mediated immunity), required for recovery from viral infections and down-regulate T helper 2 (Th2) response patterns (ex. IL-4, IL-10, and humoral immunity) (13).

a. Introduction

Th1 and Th2 response patterns must be balanced, timed, and controlled. In order to minimize, or to recover from an acute viral infection, like smallpox, the host requires: IL-12, controlled Interferon gamma (IFN γ), specific cytotoxic lymphocytes, expression of inducible nitric oxide synthase (iNOS) with increased production of nitric oxide (.NO), efficient antigen processing and presentation, ample glutathione concentrations in the antigen processing cells, and down regulation of Th2 response patterns (9,14). These factors are the major Th1 response patterns and are dependant on the glutathione concentrations of the dendritic cells, macrophages, and lymphocytes (13). Since recovery from viral infection requires Th1 cytokines, tipping the balance into Th1 responses, as with ample GSH levels, would be beneficial. During a viral infection and without sufficient GSH, the reciprocal nature between Th1 and Th2 responses leads to Th2 preponderance, increased IL-4 levels and reduction in Th1 responses (13). The clinical consequences include prolongation of the infection, increasing severity, and potential lethality.

Macrophages and the patient are at particular peril if glutathione concentrations ([GSH]) fall. For, example, in AIDS patients, the GSH deficiency in pulmonary macrophages is responsible for the unfettered growth of opportunistic microbes that are not ordinarily human pathogens, ex, pneumocystis carinii (15). HIV/AIDS is the “model” for the catastrophic consequences of diminished [GSH] (16,17).

Beyond the immediate T cell activities resulting from Th1/Th2 response patterns, the effects of IFN γ on inducing a number of additional substances is relevant in serious viral infections like smallpox, ex. (i) IFN γ fosters inducible nitric oxide synthase (iNOS), and nitric oxide (.NO) production under the protection of sufficient glutathione that serves to prevent, wasteful, dangerous reactions of NO with oxygen free radicals and lipid peroxides; (ii) IFN γ induces specific chemokines, Mig and Crg-2; (iii) activates cathepsin S, a potent cysteine protease; and (iv) causes the replacement of constitutive proteasomes with immunoproteasomes for efficient antigen processing and presentation. These four IFN γ dependent, and ultimately glutathione dependent, factors are discussed after the following paragraphs on regulation of Th1/Th2 response patterns by glutathione and NAC.

b. Thiols have Long been Recognized as Important in Th1/Th2 Response Patterns.

Jeanin et al (18), in 1995, found that N-Acetyl-L-Cysteine (NAC) and glutathione (GSH) decreased in a dose-dependent manner human IL-4 production by stimulated peripheral blood T cells and by T helper (Th) 0- and Th2-like T cell clones. This effect was associated with a decrease in IL-4 messenger RNA transcription They found that the production of IL-4-induced Ig was also reduced by these thiols, in vitro and in vivo (18).

Peterson et al (13), in 1998, found that glutathione, a regulator of intracellular redox and other aspects of cell physiology served as a key in the balance and extent of Th1 and Th2 cytokine response patterns. GSH concentrations in antigen-presenting cells were found to determine whether Th1 or Th2 response patterns predominate The basic finding was GSH up-regulated Th1 and down-regulated Th2 cytokine response patterns. One of the three experimental methods employed to deplete intracellular GSH was ethanol (13).

In March, 2001, Bengtsson et al (19) published their findings that GSH and NAC favor a Th1 response by a preferential down-regulation of IL-4 They also found these thiols down regulated the expression of CD 30, a surface antigen belonging to the TNF receptor superfamily that is expressed by activated Th cells, and is sustained in Th2 cells (19).

Dobashi et al, in May 2001 (20) published their findings that the GSH precursor, NAC, when incubated with human alveolar macrophages (AM) raised the GSH/GSSG ratio (GSSG is oxidized GSH) and enhanced IL-12 secretion from AM that had been previously exposed to lipopolysaccaride (LPS). Reciprocal loops were also shown since exposure of AM to IFN γ increased GSH/GSSG in AM cells. Further exposure of AM to IL-4 decreased GSH/GSSG in AM cells (20).

In January, 2002, Boon et al (21) showed that NAC: (i) increased influenza virus specific lymphocyte proliferation, (ii) increased IFN γ production; and (iii) enhanced specific activity of influenza specific CD8+ CTL's (cytotoxic lymphocytes) (21).

In February, 2002, Murata et al (22) published that macrophages with a high intracellular GSH concentration could be developed by in vivo application of N-acetyl-L-cysteine or glutathione monoethylester and low GSH-containing macrophages by administering L-cystine derivatives, diethyl maleate or L-buthionine-[S,R]-sulfoximine. The high-[GSH] macrophages showed elevated IL-12 and nitric oxide (.NO) production, with diminished IL-6 and IL-10 production. The low-[GSH] macrophages showed elevated IL-6 and IL-10 production and reduced .NO and IL-12 production The CD4(+) CD 44 (−) naïve T (h) 0 cells differentiated into T(h) 1 cells in the presence of high [GSH] macrophages, and into T(h) 2 with low [GSH] macrophages (22).

Giordani et al published in August, 2002 (23) that NAC down-regulated T dependent B cell activation and led to T helper cell type 1 (Th1) polarization possibly by down regulating IL-4 production (23).

c. Viral Engineering to Enhance IL-4 Expression has been a Subject of Research for at Least a Decade.

Andrew and Couper, in 1993 (24), found that laboratory-constructed recombinant vaccinia viruses expressing murine IL-4, alone, were highly toxic and poorly cleared in immunocompetent mice. This IL-4 toxicity was reversed by co-expression of IL-4 with IFN γ, wherein viral clearance occurred and there were low concentrations of IL-4 (24). This suggests that administration of high doses of GSH could overcome the increased lethality of viral engineered pox viruses enhanced with IL-4, since GSH does up-regulate Th1 cytokine production, with increased IL-12 and IFN γ, and can decrease expression of IL-4, as described in the several preceding references, above.

Sharma et al, “viral engineers” in Canberra, Australia, demonstrated in 1996 (14) that recombinant vaccinia virus (VV) engineered to express the gene for murine IL-4 was poorly cleared compared with control VV. Cytotoxic T-lymphocytes were greatly depressed during infection with IL-4 enhanced VV, compared to control VV infection, yet antiviral antibody levels and NK activity were not different between the two groups. IL-4 expressing VV caused suppression of: IL-12, IFN γ, and .NO production, which normally serves to clear viruses.

Aung et al, in 1999 (25), investigated the effect of IL-4 on Respiratory Syncytial Virus (RSV) epitope-specific CTL effectiveness. Infection by recombinant vaccinia virus (VV), constructed to co-express RSV M2 protein and IL4, compared to the VVM2 protein alone, led to decreased M2-specific CTL activity, demonstrating that local expression of IL-4, when antigen is presented, will decrease the cytolytic activity of primary and memory CD8 (+) RSV-specific CTL responses in vivo (25).

Insufficient glutathione at the time of viral infection will produce results equivalent to engineered viruses carrying extra copies of an IL-4 gene, as described above. Low [GSH] can occur as a function of age, just being over 45 years of age (26), alcohol (13), diabetes mellitus (27,28), HIV infection (15-17) and exposure to environmental toxins (29-33), among others.

d. Principal Effects of IFN γ.

A few of the principal effects of IFN γ are described here since glutathione up-regulates the Th1 response patterns, which result in increased IFN γ activities. In the face of antigen presentation, in an acute, dangerous viral infection, host recovery will be seriously compromised by a lapse, or decrease in Th1 response patterns, including these four IFN γ dependent functions:

(i) Induction of iNOS

Karupiah et al, (34) at the Laboratory of Viral Diseases, NIAID, NIH, in September 1993 demonstrated that the ability of IFN-gamma to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells' production of nitric oxide (NO)”. They also showed that IFN-γ induced NO synthase (34).

At approximately the same time, in February, 1994, Melkova and Esteban showed that INF γ inhibited the growth of vaccinia virus (VV) in a dose-dependent manner VV DNA synthesis was blocked and it correlated with increased generation of reactive nitrogen intermediates (35).

The uncontrolled induction of iNOS by IFN γ and the consequent increased production of .NO can result in a greater oxidative load and cause nitrosative stress, as studied by Noack et al (36) in 2000. However, in their studies, they attributed the resistance to toxicity of their glial cell cultures to high glutathione levels in the microglia, and to up-regulation of Mn-SOD in the mitochondria in the course of the induction of i-NOS This would relieve the total oxidative load and spare GSH.

Glutathione does have an important role in protecting NO physiology and antimicrobial properties by preventing spurious, wasteful and dangerous reactions of NO with oxygen free radicals and lipid peroxides. In short, glutathione maximizes the normal, beneficial NO functions.

(ii) Induction of Specific Chemokines

An additional function of IFN γ is the induction of specific chemokines. Mahalingam et al, in 2000, studied interferon-inducible chemokines and their antiviral effects against pox viruses (37). Monokine induced by gamma interferon (Mig) and cytokine responsive gene (Crg-2) were studied in the course of vaccinia virus and ectromelia virus infections in C57 BL/6 mice. The chemokines were induced systemically. Mig and Crg-2 induced viral clearance (37).

(iii) Activation of Cathepsin S, for Antigen Processing

Storm van's Gravesande et al, at Harvard (38), in 2002 reported on the mechanisms of up-regulation of cathepsin S activity by IFN γ. This enzyme is crucial for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells Cathepsin S is a cysteine protease and has endoproteolytic activity necessary for antigen processing (38).

(iv) Replacement of Constitutive Proteasomes, with Immunoproteasomes

IFN γ induces the replacement of constitutive proteasomes with immunoproteasomes, to direct these multiple proteases into efficient antigen processing, from proteins to antigenic peptides. Only a small sampling of the literature in this field is provided (39-43), to help delineate the far reaching effects of glutathione, and its up-regulation of Th1 response patterns.

Craiu, Akopian, Goldberg and Rock (39), from the Dana-Farber Cancer Institute, in 1997, determined that the proteasome cleaves proteins in distinct ways to help produce antigenic peptides presented on major histocompatability complex class I molecules. The proteasome determined the C terminus of the peptide whereas particular peptidases in the endoplasmic reticulum or cytosol determined the N terminus of extended peptides (39). In 1998, Beninga, Rock and Goldberg (40) showed IFN γ stimulated post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase . . . thus, IFN γ not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can (independently of proteasomes) stimulate formation of their N termini by inducing LAP (leucine amino peptidase) (40).

Meanwhile, Sijts and Kloetzel (41), in Berlin, in 2000, studied how IFN γ expressed three proteasome subunits (low molecular weight proteins, LMP2 and LMP7 as well as multicatalytic endopeptidase complex-like 1, MECL-1) to form immunoproteasomes that optimize the processing of MHC class I antigens. In the models employed, they showed that the Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope require(d) the structural features of immunoproteasomes (41).

Groettrup and colleagues, in Switzerland, built on their body of work regarding subunits and a regulatory complex of immunoproteasomes. In 2001, Khan . . . and Groettrup (42) showed that during an immune response to lymphocytic chroriomeningitis virus or the bacterium Listeria monocytogenes, immunoproteasomes largely replace(d) constitutive proteasomes . . . in the liver (42).

In 2002, Goldberg and Rock, out of Harvard (43), reviewed The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides (43).

Glutathione concentrations in pivotal cells such as monocytes/macrophages, dendritic cells, and lymphocytes are vulnerable and decline rapidly in response to alcohol, toxins, oxidative stresses, physical/emotional stress, infections, trauma, burns, and non-bacterial and bacterial sepsis. The Th1/Th2 balance shifts to Th2 predominance as a result, making recoveries difficult.

Examples of dangerous viruses, wherein [GSH] and shifts in Th1/Th2 have been shown to be important with regard to parameters of desirable host defenses and clearance of viruses, include vaccinia virus, recombinant VV expressing IL-4, Respiratory Syncytial Virus, ectromelia, and herpes simplex-1.

2. A principal property of glutathione (GSH) is maintenance of the Redox Potential, i.e. the Reducing vs. the Oxidizing Potential [GSH]/[GSSG]. This ratio is in the range of 500. The normal [GSH] in cells is 5-10 mM.

a. Introduction

This thiol is a major determinant of the Reduction Oxidation Potential in the cell and is protectively involved in diverse cell activities, including Control of cell cycle progression in human natural killer cells (44), and defensive responses to infections (15-17,21), to chemical exposures (29,31), and to other detrimental factors such as diesel exhaust particles (32,33), aging (26,46), diabetes (27,28), and photo-oxidative retinal damage (45). As noted by the CDC (11), physiologic host factors make the difference in a case (of smallpox) and how severe it will be (11). The effects of glutathione concentrations, [GSH], on Redox and the consequent effects on specific entities such as the NFκB family, TNFα, cytokines, COX-2 and adhesion molecules, provide substance for the term, host factors, and also provide direction for additional therapeutics, for example, raising [GSH] and simultaneously protecting the patient from chemical toxins and other factors detrimental to [GSH], as cited previously and below.

Biochemical Evolution proceeded towards a stable/controllable range of pH, pO₂, osmolarity, and [Na⁺]/[K⁺]; so too has this process led to a stable/controllable Redox. When the concentration of GSH is high, Redox is high. Then, it can control and slow the excess activation of the NFκB family, oxidant-sensitive transcription regulator(s) (47); of proinflammatory cytokines (48); COX-2 (47); adhesion molecules (49); and TNFα and IL-1β that cause secondary cascades (50,51). A significant decrease in GSH results in a decline in Redox and activation of the NFκB family and the other factors.

b. Prevalence of GSH Insufficiency and Compromised Redox

The consequences of a low [GSH] and low Redox, as occur in other viral infections such as HIV, and in diabetes mellitus, and other disorders, are discussed and referenced here, and in detail in Section 9 of this application. These are examples in humans, and in laboratory animals, of the adverse consequences of GSH insufficiency, and excessive the compromised Redox that follows.

The destructive effects on [GSH] and Redox: by sulfur mustard (52); by the “sepsis” syndromes, (53); destructive effects by diesel exhaust particles (32,33); by other infections (15-17,21); and other pollutants (29), have relevance to: (i) the battlefield; (ii) the reconstruction of liberated countries wherein injuries, pollution and “booby” traps threaten our military and the indigenous population; and (iii) urban terrorist attacks;

In a recent review, for example, Karol et al in 2001 published a large body of data on the effects of chemical toxins in the work place, including the Documentation of Threshold Limit Values (TLV's) published by the American Congress of Governmental Industrial Hygienists, and the results of computer-generated structure-activity relationship models based on physicochemical properties of the agents and their stoichiometric reactions with glutathione. The results indicate Their in vivo reactions with thiols may result in glutathione deficiency with consequent alteration in cellular reduction-oxidation (redox) status, release of cytokines, and promotion of T helper cell 2 phenotype (29). While this publication from the University of Pittsburgh School of Public Health was concerned with Occupational Health and interdicting chronic diseases that are in part, based phenotypically in Th2 cell types, there are additional applications to treating patients (military and civilian) who already have the disorders described by Karol et al, and the post war disorders, if in fact they are linked to chemical exposures.

The CDC, logically and experientially, has stated (11) that physiologic host factors make the difference in a case (of smallpox) and how severe it will be has applications in managing smallpox infections that may occur among the several million people who have predominant Th2 problems, an immunologic state known to be associated with glutathione insufficiency and low redox. Smallpox infection may be more serious among individuals who start off being glutathione insufficient and only marginally able to produce IL-12, and IFN γ, and to marshal smallpox-specific cytotoxic lymphocytes. It is possible that GSH insufficiency and predominance of T helper cell 2 phenotype (29) represent factors in common among the seemingly disparate groups who face increased risks of complications if vaccinated vs. smallpox with the present live vaccine.

On this note of compromised glutathione concentrations and diminished redox status in humans, the recent publication in 2002 by medical science leaders in glutathione, from Emory University, DP Jones and P Sternberg, revealed a precipitous decline in GSH/GSSG redox beyond 45 years of age (26). While there was a linear oxidation of cysteine/cystine redox state with age at a rate of 0.16 mV/year over the entire age span (in plasma) . . . GSH/GSSG redox was not oxidized prior to 45 years and subsequently was oxidized at a nearly linear rate of 0.7 mV/year This amounts to four times greater oxidation of GSH after 45, compared to cysteine (26).

A “companion” publication by Murata et al in 2002 (46) linked GSH/GSSG ratios in peritoneal resident macrophages, in thioredoxin transgenic mice, compared with age matched wild type (WT) mice with: (i) intracellular redox status, and (ii) sustained maintenance of Th1 prevalence during aging Their measurements of GSH/GSSG, IFN γ, IL-10 and IL-4 were expressed as IFN γ/IL-10 and IFN γ/IL-4 ratios (46). These authors indicate an advantageous delay in the onset of autoimmune disease among the mice with higher ratios of GSH/GSSG, INF γ/IL-10 and IFN γ/IL-4, comparing the transgenic mice with sustained maintenance of Th1 prevalence during aging with WT mice (46).

c. Thiols, Natural Killer Cells, and Innate Immunity

In dealing with lethal, acute viral infections it is important to consider not only acquired immunity through T-cell responses, but also innate immunity via NK cells.

The preceding discussions regarding the benefits of high concentrations of glutathione, [GSH], Redox, and host responses involving T cells and acquired immunity have some applications to innate immunity and NK cells as well. In 1997, Yamauchi and Bloom (44), from the Center for Biologics Evaluation and Research, FDA, studied the importance of thiols in NK responses to IL-2. They had demonstrated that NK responses to IL-2 were redox regulated.

Their additional work showed that the IL-2-dependent cell line, NK3.3 . . . neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2 (44).

d. NFκB and its Dependent Gene Networks

NFκB is at the epicenter of global cellular responses after viral infection (54), as summarized by Tian . . . Brasier and colleagues from the University of Texas, in 2002. Using high-density oligonucleotide microarrays, they investigated the breadth of genes induced by Respiratory syncytial virus (RSV) infection that were dependent on NFκB. They used cells stimulated with TNFα, to activate NFκB, as well as RSV infection, the replication of which also activates NFκB. They concluded that NFκB was required for RSV-inducible expression (of) chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins Among the latter were complement components and growth factor regulators (54). They concluded that N-κB action induces global cellular responses after viral infection (54).

e. Molecular Mechanisms of GSH Insufficiency and Oxidative Stress in Gene Signaling

Rahman et al, in 2002, out of the University of Edinburgh (48) provided a molecular mechanism for the pro-inflammatory effects of oxidative stress (48). In order for transcription factors in gene signaling networks to bind DNA, unwinding of DNA is necessary to provide access. Acetylation/deacetylation of histone residues on the histone core around which DNA is coiled (48) is required for such access.

Rahman and colleagues exposed a line of human alveolar epithelial cells, A549, to oxidative stress by H₂O₂ and TNFα, which led to glutathione oxidation (GSSG), and depletion of the reduced form of glutathione, GSH, in the presence or absence of TSA, a substance that inhibits deacetylation of histone residues and the unwinding of DNA.

The deacetylation is a reversible process and is controlled by acetyl transferases and deacetylases. TSA inhibits deacetylases and increases acetylation of histone proteins. While H₂O₂ and TNFα enhanced DNA binding of NFκB and AP-1, the addition of TSA potentiated the increased AP-1 and NFκB binding and also increased IL-8 release beyond the effects of H₂O₂ and TNFα. They concluded that the oxidant H₂O₂ and the proinflammatory mediator, TNF-α, induce histone acetylation which is associated with decreased GSH levels and increased AP-1 and NF-κB activation leading to enhanced proinflammatory IL-8 release in alveolar epithelial cells. This indicates a mechanism for the proinflammatory effects of oxidative stress (48).

f. Viral Infections, NFκB, Thiol Redox and Pro-Inflammatory Responses

Armed with the references of: (i) glutathione enhancing NK cell responses and their progression through the cell cycle (44); (ii) glutathione inhibiting the oxidant-sensitive transcription factor NFκB (47); (iii) NFκB at the epicenter of global cellular responses after viral infection regulating chemokines, transcriptional regulators and other factors (54); and Rahman and colleagues (48) who provided a molecular mechanism for the pro-inflammatory effects of oxidative stress involving the unwinding of DNA from its histone core (48), it becomes possible to better understand [GSH], and thiol Redox in host responses and defenses as in the following sampling of fifteen references, (i-xv), three of which relate to HIV infection.

(i) Pharmacologic Thiols, NFκB, and TNF α

In 1996, Neuschwander-Tetri and associates (55) found that Oxidant stress up-regulates and antioxidants down-regulate the pleiotropic transcription factor NFκB, a DNA binding protein that induces expression of cytokines and vascular adhesion molecules . . . (and) increased cellular glutathione levels blocked NF-κB activation and inhibited the release of TNF-α (55).

(ii) Activation of Pro Viral HIV DNA by NFκB and TNF α

Baruchel and Wainberg were among the early investigators in this area, in 1992 (15), in their paper entitled, The role of oxidative stress in disease progression in individuals infected by the immunodeficiency virus, state:

“ . . . This review describes the potential role of oxidative stress as a cofactor of disease progression from asymptomatic human immunodeficiency virus (HIV) infection to the acquired immunodeficiency syndrome (AIDS). Oxidative stress is a known activator of HIV replication in vitro through the activation of . . . NF-κB, which in turn stimulates HIV gene expression . . . Tumor necrosis factor alpha (TNF-α) . . . is also involved in the activation of HIV infection through similar mechanisms. TNF-mediated cytotoxicity of cells exposed to this substance is related to the generation of intracellular hydroxyl radicals . . . In favor of the role of oxidative stress in HIV . . . progression is the consumption of glutathione (GSH), a major intracellular antioxidant, during HIV infection and progression. GSH is known to play a major role in regulation of T cell immune functions. Oxidative stress may also play an important role in the genesis of cellular DNA damage and, in this context, may be related to HIV-associated malignancies and disease progression. The role of antioxidants as components of therapeutic strategies to combat HIV disease progression is discussed . . . . ”

(iii) Antioxidants and HIV

Lee, Beauparlant, Elfond et al built on this area with the publication of their work in 1997 (16), Selective inhibition of I-κB alpha phosphorylation and HIV-1 LTR-directed gene expression by novel antioxidant compounds, which states:

Oxidative stress activates the NFκB/Rel transcription factors which are involved in the activation of numerous immunoregulatory genes and the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). In the present study, we examined the effects of established and novel compounds including antioxidants, ribonucleotide reductase inhibitors, and iron chelators on NFκB activation and HIV LTR-mediated gene expression induced by TNF-α. N-Acetylcysteine (NAC), pyrrolidinedithiocarbamate (PDTC), and Trimidox (TD) at various concentrations inhibited TNF-α-induced NFκB binding in Jurkat cells. Pretreatment of cells with these compounds prior to stimulation prevented I κB alpha degradation. Phosphorylation of I κB alpha, a prerequisite for its signal-induced degradation, was abrogated in these cells, indicating that oxidative stress is an essential step in the NFκB activation pathway . . . Synergistic induction of HIV-1 LTR-mediated gene expression by TNF-α and the HIV-1 transactivator Tat in Jurkat cells was significantly suppressed in the presence of NAC and TD, but not PDTC

(i) GSH Protects Vs. Cytomegalovirus

Immunocompromised patients often have cytomegalovirus induced vascular pathology. Vossen et al suggested, in 1997, (17) in their publication that a high thiol redox status in endothelial cells might provide a barrier to CMV, state:

A relation between viral infection and oxidative stress has been recognized for human immunodeficiency virus and herpes simplex virus-1 infections, but little is known in this respect for CMV infections . . . this study suggests that a high endogenous thiol redox status may contribute to the apparent barrier function of endothelial cells with respect to CMV infection and that oxidative stress may facilitate CMV infection of the vascular wall

(ii) IL-18 Stimulation of IFN γ is GSH Dependent

Murata et al, in 2002, (56), found that the ability of IL-18 to “strikingly augment the production of IFN-gamma in mature splenic dendritic cells treated with IL-12 was “ablated when glutathione intracellular concentrations were decreased (56). This same effect of “intracellular glutathione deprivation was seen in macrophages.

(iii) Thiols and Inhibition of COX-2 Expression

Kim and associates, in 2002, (47), showed the H. pylori infection activated NFκB and COX-2, and that both were inhibited with glutathione, NAC, and pyrrolidine dithiocarbamate, also a thiol antioxidant. In conclusion, oxidant-sensitive transcription factor NF-κB may play a novel role in expression of COX-2 by H. pylori stimulation in gastric cancer cells (47).

(vii and viii) Chemokines

With regard to chemokines (50) which are important for the progress of an inflammatory response by the recruitment of immuno-competent cells (51), Blackwell et al, in 1996, (50) found that treatment of rats with the thiol antioxidant, NAC, decreased NFκB activation by LPS, and sufficiently diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue to decrease lung lavage neutrophil count six fold (50). Ciesielski and associates, in 2002, (51), found that TNF α-induced macrophage (CXC) chemokines secretion is more dependent on NFκB expression than lipopolysaccharide-induced macrophage (CC) chemokines secretion (51).

(ix, x, xi, xii) Adhesion Molecules

Adhesion molecules, which are instrumental in leucocyte-vessel wall interactions, which are involved in variola-infected mononuclear cells adhering to vessels in the dermis, preparatory to their egress into the skin (3), are redox regulated, as shown in these four publications. Lee et al, in 2003, (57), demonstrated the Redox-regulated mechanisms of IL-4-induced MCP-1 (monocyte chemoattractant protein-1) expression in human vascular endothelial cells (57). They employed NAC and pyrrolidine dithiocarbamate (PDTC). IL-4 induced MCP-1 gene expression by dramatically increas(ing) the transcription factor, signal transducers and activators of transcription 1 (STAT1) DNA binding activity, an effect that was attenuated by PDTC (57). Lee and colleagues had earlier shown, in 2001 (58) that IL-4 up-regulated vascular cell adhesion molecule-1 (VCAM-1) by inducing oxidative stress. Xia et al, in 1998 (59) demonstrated that the antioxidant PDTC or sodium salicylate inhibited IL-4 induced expression of P-selectin, an adhesion molecule elaborated by human endothelial cells. Ikeda and associates in 1994 (60) published their findings on the “Suppressive effect of antioxidants on intercellular adhesion molecule-1 (ICAM-1) expression in human epidermal keratinocytes . . . ICAM-1 is strongly expressed by human epidermal keratinocytes during the course of inflammatory skin disease (60). See the 5 Scanning Electron Micrographs in Section 3, pp. 57-61.

(xiii, xiv, xv) Matrix Metalloproteinases

After Variola infected mononuclear cells adhere to the dermal vessels (3), their egress through the endothelial pavement and through the dermal collagen, and epidermal cells is most likely enhanced, like that of other migratory human monocyte-dendritic cells (61), and invasive cancer cells (62), by matrix metalloproteinases (MMP's). These are zinc-ion containing endopeptidases that are Ca⁺²-dependent and currently constitute a family of 21 MMP's. They are countered by tissue inhibitors of metalloproteinases (TIMP).

Osman and colleagues, in 2002 (61), studied the migratory behavior of dendritic cells (CD's) derived from human monocytes through an in vitro model of extra cellular matrix, the Matrigel trans-well migration assay. Immature DC's were derived with IL-4/granulocyte macrophage-colony-stimulating factor (GC-CSF), and mature DC's were induced by TNFα and modified vaccinia virus Ankra (MVA). The ‘mature’ DC's (induced by TNFα and MVA) showed an increased migration through Matrigel, which was significantly inhibited by inhibitors of matrix metalloproteinases (MMP) (61). Presumably, curbing TNFα expression with glutathione may inhibit MMP activities.

Esteve et al, in 2002 (62) studied gene signaling leading to MMP-9 expression by inflammatory cytokines, II-1 and TNFα. They showed that a particular protein kinase C isoform (PKC-zeta) was induced by IL-1 and TNFα, and blocking PKC-zeta activity abolished MMP-9 activity and gene expression induced by IL-1 or TNF-α. These processes were shown to be NF-κB dependent and were completely prevented by NF-κB-binding site mutation in the mmp-9 promoter (62).

A study by Uemura et al in Circulation Research, in 2001 (63), found that MMM-9 expression in vascular endothelial cells was redox sensitive, and that the oxidative stress secondary to hyperglycemia provided a rationale to use antioxidants to suppress MMM-9 expression (63). TNFα, as employed by Osman and colleagues (61), and by Esteve et al (62) induces oxidative stress, as does the hyperglycemia noted by Uemura et al (63). Pharmaceutical GSH may be beneficial in this context.

This concludes a review of the second property of glutathione and Redox. When [GSH] declines, Redox falls, resulting in NFκB activation, acetylation of histone residues and unwinding of DNA that enhances access by NFκB and other transcriptional regulators translocated to the nucleus, followed by activation and expression of proinflammatory cytokines, chemokines, COX-2, and secondary cascades that follow the transcription regulators and the increased TNFα production.

Examples of viral infections, wherein progression of the infection and cumulative tissue toxicities are related to declines in [GSH] and Redox, include HIV/AIDS, and Respiratory Syncytial Virus infection.

3. The biochemical neutralizing reactions of glutathione vs. reactive intermediates are grouped arbitrarily into three categories for purposes of discussion:

a. Detoxify Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) That Otherwise Damage Cell Membrane Lipids, Proteins and Nucleic Acids and Result in Cellular and Tissue Toxicities

b. GSH Protects Mitochondria vs. H₂O₂, Bioenergetic Failure, and Exaggerated, Apoptotic Processes That Add to Cumulative Tissue Toxicities

c. GSH and It's Companion Enzymes Prevent Excessive, Uncontrolled, Tissue-Disrupting-Oxidations of Arachidonic Acid (lipid hydroperoxides, schism of peroxides, alkoxy radicals, hydroxyl radicals, isoeicosanoids and eicosanoids)

Several viral infections provide examples of the ameliorative importance of intracellular glutathione concentrations, despite specific differences in the diseases, attributable to differences in viral proteins, such as variola proteins that permit early evasion of host defenses, differences in the cells affected, replicatory processes, and host factors. The viruses eventually do encounter: (i) the human immune system; (ii) clearance mechanisms; and (iii) varying measures of inflammatory reactions. Immunologic encounters and microbial clearance mechanisms, in general, involve phagocytosis with ROS, and clearance by RNS and by apoptosis, while cytokines, such as TNFα, IL-4 and IFN γ evoke ROS and RNS, directly and indirectly, via signaling pathways; for example, IL-4 up-regulates 12/15 lipoxygenases and down-regulates GSH peroxidases to create extensive, lipid hydroperoxide-derived ROS, as described further on.

The continuous intracellular concentrations of glutathione within: (i) the responding immune system cells; (ii) in the cells directly affected, and (iii) in by-stander cells can make a difference. The uncontrolled, combined presence of ROS, RNS, pro inflammatory cytokines, and reactive by-products of arachidonic acid “metabolism” result in “severe inflammatory response syndrome” that is found in non bacterial sepsis states, and in serious viral infections such as smallpox, and influenza epidemics. The molecular pathologic processes are qualitatively shared.

a. Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), Glutathione, and Molecular Pathology.

ROS and RNS cause substantial, cumulative tissue toxicities, particularly because they initiate rapid chain reactions that yield additional, toxic by-products. ROS and RNS are discussed separately because much of the cited literature separates them, a consequence of awareness, focus, and funding sources. The activated monocytes/macrophages, for example, actually produce both, via their 15-lipoxygenases (ROS), and the induction of iNOS (RNS) by IFN γ, generally within a similar time frame. As a result, a variety of complex interactions occur and yield reactive nitrogen species, such as peroxynitrites, that are more injurious than the initial nitric oxide. The separation is artificial in the following subsections, but provides a degree of clarity.

(i) ROS Mediated Tissue Toxicities and Molecular Pathology in Viral Infections and GSH Protective Effects

The Human Immunodeficiency Virus (HIV) was among the early viruses studied wherein ROS, also termed “free radicals”, were found to be significant factors in the infection, its molecular pathology, and progression, as described in the references that follow.

Buhl and associates in 1989, in Lancet (64) demonstrated in symptom free HIV-seropositive individuals significant losses of glutathione Total and reduced glutathione concentrations in the plasma . . . were about 30% of those in the normal individuals (and in the lung epithelial lining fluid, ELF) about 60% of those in the controls (64). They concluded that glutathione immune deficiency may contribute to the progressive immune dysfunction of HIV infection (64).

Wahl and co-workers in 1989, in the Proceedings of the National Academy of Sciences (U.S.A.) (65) found that an HIV surface glycoprotein, purified gp120, was able to induce “monocyte arachidonic acid metabolites and Interleukin 1 (65). Studies on gp120, referenced further on in 1996, in this subsection, show this occurred via a signaling pathway, and not by direct oxidation.

The direct oxidation of polyunsaturates, like arachidonic acid, requires reactive oxygen species as shown by Bielski et al in 1983 in “A study of the reactivity of HO₂/O₂— with unsaturated fatty acids” (66). Bielski and others helped confirm the earlier body of work by H. B. Demopoulos and colleagues dealing with lipids and free radicals in the major central nervous system disorders (67-70), in carcinogenesis (71), and other disorders (72). In these seminal studies, data was provided for the first time in controlled, timed studies that free radicals were generated by the pathologic process, which then attacked polyunsaturates like arachidonate, causing chain reactions that consumed tissue antioxidants, and left a pathologic trail of cellular and tissue injuries.

Further research by Esterbauer and co-workers (73) in 1991 and by Gutteridge (74) in 1995 have helped to establish the concept that the finding of lipid peroxides, or lipid peroxidation products, or losses of tissue antioxidants, such as glutathione, are valid indications/biomarkers of tissue damage by uncontrolled, destructive free radical reactions now termed “oxidative stress”.

Uhlson et al (75), in 2002, provide an example of the disruptive effects of oxidative stress, by ozone, on macrophages and pulmonary tissues that are relevant for understanding the viral research cited in oxidative stress in support of adjunctive GSH therapy for smallpox threats and therapy. Low levels of ozone exposures of 62.5, 125 and 250 ppb of unsaturated fatty acyl groups in pulmonary surfactant phospholipids resulted in oxidative stress, as expected, and yielded a biologically active product in that it reduced elicited macrophage viability by necrosis (75). This damaging, oxidized phospholipid, 1-palmitoyl-2-(9¹-oxo-nonanoyl)-glycerophosphocholine, not only decreased macrophage viability, but also induced apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity (75).

Oxidative stress and glutathione insufficiency have been substantiated in HIV infection, along with the cumulative tissue toxicities that ensue, and the progression to AIDS. Eck . . . and Droge (76) in 1989 found low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1-infected patients In 1991, Kalebic . . . Meister and Fauci (77) conducted definitive research that was stimulated, in part, by Buhl (64), Wahl (65), and Droge (76), but largely by the expertise and intellectual drive provided by Meister and Fauci. “ . . . The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13 acetate (PMA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-α, or IL-6, in the absence of cytotoxic or cytostatic effects. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection . . . . ” Attempts, however, by pharmaceutical entities to deploy thiols vs. HIV/AIDS did not meet with success because GSH at that time was not yet bioavailable.

Droge and co-workers, in 1992 correlated the thiol deficiency induced by HIV with T-cell dysfunction and advocated treatment with N-acetylcysteine (78). Droge, six years earlier, in 1986 (79) had discovered that glutathione augments the activation of cytotoxic lymphocytes in vivo (79). Baruchel and Wainberg (15), in 1992 reviewed this area of “oxidative stress as a co-factor of disease progression (15). W. Z. Ho and S. D. Douglas (80) in 1992 added the finding that HIV-1 expression in peripheral blood mononuclear cells (PBMC) was blocked or substantially reduced by GSH . . . This anti-HIV-1 effect persisted in these cultures for at least 35 days without evidence of significant increase in HIV-1 expression. Thus, a single pulse exposure of HIV-1-infected monocyte/macrophages with GSH led to a sustained, concentration-dependent decrease in HIV-1 p24 antigen levels, as well as, reverse transcriptase activity without producing detectable cellular toxicity in monocyte/macrophages

Sandstrom and colleagues (81) in 1994 studied a cell line, 8E5, that is chronically HIV-infected and also has a marked reduction in glutathione-peroxidase activity They reported that HIV gene expression . . . renders 8E5 cells 10-fold more sensitive to killing by 15-hydroperoxyeicosatetraenoic (15-HPETE), as well as several other hydroperoxy fatty acids because the low GSH-peroxidase activity in 8E5 cells prevented the conversion of the toxic 15-HPETE (15-LOOH) to the indolent 15-hydroxy-eicosatetraenoic acid 15-HETE (15-LOH) (81). This study emphasizes the importance of ancillary glutathione enzymes that provide added protection against the toxic 15-HPETE that can undergo peroxide schism [15-LOOH→15-LO.+.OH] and start free radical chain reactions via the alkoxy radical (LO.) and the hydroxyl radical (.OH). Pre-emptive conversion to 15-HETE by glutathione peroxidases avoids this (81).

The lipid-peroxidizing enzymes, like 12/15-lipoxygenases and the hydroperoxyl lipid-reducing enzymes, like GSH-peroxidase, are inversely regulated by interleukins 4 and 13 (82). These two interleukins up-regulate the 12/15 lipoxygenases and down-regulate the phospholipid hydroxoperoxide GSH-peroxidases according to Schnurr et colleagues in their 1999 publication (82).

The net result is accumulation of dangerous lipid hydroperoxides (LOOH) that can start ROS pathology. This helps explain the observations in the older literature that IL-4 was a pro-oxidant that initiated free radical pathology.

Shatrous and associates (83) in 1996 confirmed the complex role of HIV gp120 reported previously by Wahl (65). Shatrous and the “mentor”, W. Droge (83), demonstrated that gp120 amplified the activity of TNFα with regard to free radical generation. This in turn activated NM, which translocates to the nucleus and activates HIV proviral DNA by attaching to binding sites on the long terminal repeat (LTR). This results in rapid replication of HIV. The gp120 protein exerts its effects on GSH reduction through the p56 1ck protein tyrosine kinase, which “transmits” a signal that increases free radical, oxidative stress (83).

Additional ROS, GSH publications regarding HIV include, for example: Sung J H et al. Protective effect of glutathione in HIV-1 lytic peptide 1-induced cell death in human neuronal cells. J Neurovirol. 2001; 7:454-65 (84).; Muller F et al. Virologic and immunologic effects of antioxidant treatment in patients with HIV infection. EuriClin Invest. 2000; 30:905-14. (85).; Choi J et al. Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice. J. Biol Chem. 2000; 275:3693-8. (86).; Walmsley S L et al. Oxidative stress and thiol depletion in plasma and peripheral blood lymphocytes from HIV-infected patients: toxicological and pathological implications. AIDS. 1997; 11:1689-97 (87).

Viral Hepatitis, particularly Hepatitis C, is characterized, in part by oxidative stress that causes significant, cumulative tissue toxicities as observed in some viral infections. In the case of HCV, ROS are generally produced secondarily, for example, via signaling pathways.

Zhu and colleagues (88), in 1998 demonstrated that the hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1. This leads to ROS generation, which contributes to patient morbidity, and if apoptosis follows, it may contribute to further liver necrosis and mortality.

Park and co-workers (89), in 2001, found that HCV core protein activated c-Jun N-Terminal Kinase through the TRADD and TRAF 2 signaling complex, resulting in oxidative stress.

Vendemilae et al (90), in 2001, described oxidative stress in symptom-free HCV patients, while Moriya and colleagues (91), in 2001, found evidence of oxidative stress, without inflammation in a mouse model.

Floyd R A, a long-standing basic investigator in oxidative stress and related free radicals, demonstrated an association between ROS and HCV disease activity in 1996 (92). There are, apparently, non inflammatory sources for oxidative stress in HCV infection including a high content of lipid hydroperoxides in HCV patients (93). Further, Barbaro and colleagues (94), and others (95), described ferritin and hepatic glutathione abnormalities in chronic HCV patients. Iron, particularly organic iron, is a potent catalyst for lipid peroxidation and other free radical chain reactions (95).

The lasting “imprint” of another productive ROS researcher, Fridovich at Duke, may have a relationship to the publication by Suliman et al (96) in 2001 regarding influenza. Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules may exacerbate lung injury after influenza virus pneumonia. We hypothesized that enhanced expression of extracellular superoxide dismutase (EC SOD) in mouse airways would attenuate the pathological effects of influenza pneumonia. We compared the pathogenic effects of a nonlethal primary infection with mouse-adapted Hong Kong influenza A/68 virus in transgenic (TG) EC SOD mice versus non-TG (wild-type) littermates. Compared with wild-type mice, EC SOD TG mice showed less lung injury and inflammation as measured by significant blunting of interferon-gamma induction, reduced cell count and total protein in bronchoalveolar lavage fluid, reduced levels of lung nitrite/nitrate nitrotyrosine, and markedly reduced lung pathology. These results demonstrate that enhancing EC SOD in the conducting and distal airways of the lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.

(ii) RNS Mediated Tissue Toxicities and Molecular Pathology in Viral Infections and GSH Protective Effects

Nitric Oxide is a reactive free radical, with a half-life in vivo (about 10 seconds) that is slightly longer than the more reactive free radicals, superoxide anion (.O₂—) and hydroxyl (.OH). Nitric oxide shares solubility characteristics with molecular oxygen and is somewhat non polar (69-71, 97). Molecular oxygen is approximately seven times more soluble within the non polar milieu of the hydrophobic midzone of cell membranes, placing .NO and O₂ at the most sensitive, oxidizable areas of the fatty acyl chains of membrane phospholipids, the unsaturated bonds (69-71, 97). Radical reactions in these non polar membrane midzones (alkoxy LO. and mobile .OH) adversely affect not only the lipids, but also the hydrophobic segments of membrane proteins. This can lead to inactivation of enzymes and alterations of other membrane macromolecular complexes (69-71).

The relatively non polar structure of nitric oxide, and its slightly less reactive properties, compared to superoxide and hydroxyl free radicals, provides nitric oxide with a “range” of approximately 500 μm or more since it diffuses at 50 μm/second and has a half-life, in vivo, estimated at 10 seconds (97). The practical tissue and molecular pathologic consequence of this range is the effect on by-stander cells, each one averaging 15-25 μm in diameter in a tissue (lymph nodes, skin, etc.) containing activated monocyte/macrophages, that have just arrived via diapedesis, carrying smallpox or other viruses, and elaborating nitric oxide, lipid hydroperoxides, and cytokines, among other substances.

Nitric oxide reacts with superoxide and forms a potent oxidant, peroxynitrite (⁻OONO) (98). The disadvantages of this reaction are two-fold: (i) the desirable anti microbial effect of NO is lost, and (ii) a spectrum of biological molecules are damaged by indiscriminate oxidations. A stable oxidation end product that is increasingly recognized as an indicator/biomarker of oxidative stress is Nitrotyrosine, now viewed as a distinctive ‘molecular fingerprint’ for nitric oxide-derived oxidants (99). Nitric oxide also reacts with thiols, generally by first forming N₂O₃, which has more direct nitrosating properties, and readily forms S-nitrosothiols (100).

Nitrosylation or nitrosation of proteins is a prevalent reaction that can involve additional nucleophilic side chains, in addition to sulfhydryl groups, such as hydroxyls, amines and aromatic carbons (101). Stamler and his colleagues at the Brigham (101), in 1996, examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules . . . nitrosation reactions (were) sustained at sulfur, oxygen, nitrogen and aromatic carbon centers (101). Stamler et al (101) found thiols were the most reactive, and pre-nitrosation reduction of protein tertiary structure resulted in polynitrosylation, as more free thiols were exposed by the pre-nitrosation reduction. While some polynitrosylated proteins, like bovine serum albumin (BSA), exhibited physiologic nitric oxide properties such as vasodilator and anti-platelet activities, nitrosylation of enzymes generally had an adverse effect on enzymatic activities. Stamler's group recognized the implications of nitrosating tyrosine residues in enzymes and the potential disruption to phosphorylation-dependent processes (101).

While nitrosation of enzymes and formation of S-nitro groups at their active centers was found to be potentially reversible with thiol agents that restored activity (102), the nitrosation of other proteins, and in particular nitrotyrosine formation, is pathogenic (99).

The inflammatory reactions engendered by monocytes/macrophages, although studied within atherosclerotic arteries (99), provide valuable information on RNS pathology for inflammation elsewhere. In many instances, the basic molecular pathologic processes are similar.

The Vita, Hazen group (99) emphasized, in addition, the pathology resulting from superoxide excesses arising from leucocyte-derived NAD(P)H oxidase complexes, and the simultaneous consumption of nitric oxide by leucocyte-derived myeloperoxidase. The net result was compromised NO physiologic effects by this simultaneous, dual action of: (i) removing NO by the formation of peroxynitrite, and (ii) the consumption of NO by myeloperoxidase activity (99). This same “mix’ of activated leucocytes, with their NAD(P)H oxidase complexes, and myeloperoxidase, plus activated monocytes/macrophages elaborating nitric oxide, is found at sites of inflammation and infection, as in smallpox lesions (2,3).

The formation of Nitrotyrosine, as studied by the Vita, Hazen group (99), has important implications for the inhibition of pox virus DNA topoisomerase discussed and referenced, further on, under the fourth property of glutathione.

In addition to glutathione affecting the thiol reactions of these topoisomerase sulfhydryl enzymes, there is a critical Tyrosine-274 residue in vaccinia topoisomerase. As will be detailed further on under property number four, this tyrosine forms a 3′-phosphodiester bond with the DNA “backbone”. If nitric oxide attacks it (Stamler's group demonstrated that protein hydroxyls come under attack, along with sulfhydryls and other moieties [101]), then the resulting nitrotyrosine could effectively convert the topoisomerase into an endonuclease forming a 60 bp duplex DNA with a 3′ phosphate terminated product. This potential, to create a crippling nitrotyrosine within a critical site in a pox virus DNA topoisomerase, may be practical as discussed under property number four, particularly if ample glutathione is present to prevent wasteful, and harmful formation of peroxynitrites that prevent the useful functions of nitric oxide. One of the hypotheses described further on considers that glutathione can reduce functional disulfide formation and reduce inter- and intra-chain disulfides in topoisomerase, thereby making cysteines and the Tyr-274 accessible to nitrosating reactions. As Stamler showed (101), nitrosation of proteins involves nucleophilic side chains such as sulfhydryls and hydroxyls. Nitrosations and glutathionylations can be selective versus pox virus topoisomerases, because it is the monocyte/macrophages/dendritic cell population that harbors much of the viral load, and it is this cell population that generates significant quantities of nitric oxide. In the presence of sufficient glutathione, ROS and RNS can be curtailed to permit orderly formation of S-nitrosoglutathione, a long lasting, diffusible NO donor that would be available and in close, intracellular proximity to the NO-sensitive hydroxyl of Tyr-274 of pox virus DNA topoisomerase. Further details are provided under property number 4 of GSH.

Sharma (14) and other pox viral “engineers”, like Andrew and Couper (24) demonstrated that IL-4 enhancement of vaccinia virus suppressed the IFN γ-NO— mediated clearance of virus. Diminishing the morbidity and mortality of smallpox infections requires the enhancement and preservation of the anti-viral, microbial clearance properties of nitric oxide, but without the RNS-mediated pathology. This was recognized “early on” by Karupiah et al in 1993 at NIAID (34), regarding their IFN γ/NO studies with ectromelia, vaccinia and herpes simplex-1 viruses in mouse macrophages. Ample, consistent concentrations of intracellular glutathione do provide the balance, potency, and safety required to prevent excessive ROS/RNS reactions that otherwise deplete glutathione, and then lead to perpetually worsening tissue toxicities. This is discussed and referenced in the next subsection.

In a publication entitled “The protective role of thiols against nitric oxide-mediated cytotoxicity in murine macrophage J774 cells”, in 1997, Zamora and colleagues (103) demonstrated that the viability of these macrophages was diminished, under their experimental conditions, by exposure to an NO-releasing compound, S-nitroso-N-acetyl penicillamine (SNAP). When the glutathione concentration was experimentally reduced in these macrophages, cell viability was decreased further. However, the macrophages were protected when the thiol, N-acetylcysteine, was used as a pretreatment (103).

In 1998, Arstall and co-workers (104) in the Cardiovascular Division at Brigham and Women's Hospital, showed that increasing GSH (glutathione) levels attenuated the decline in myocyte CK (Creatine Kinase) activity following exposure to the NO donor, S-nitroso-N-acetylcysteine (SNAC). On the other hand, when GSH levels in the myocytes were decreased, the S-nitrosation of creatine kinase by SNAC was enhanced and enzyme activity diminished.

Cuzzocrea and colleagues, in 1998 (105), studied the cytotoxic effects of peroxynitrite in human venous endothelial cells and in rat aortic smooth muscle cells. They found that glutathione attenuated the peroxynitrite . . . induced suppression of mitochondrial respiration . . . formation of nitrotyrosine . . . protein oxidation . . . DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthase When GSH concentrations were lowered experimentally, the peroxynitrite-induced cell toxicities worsened (105).

The synergistic toxicity of NO and ROS by forming peroxynitrite and other cytotoxic agents, was found to be amenable to inhibition by antioxidant enzymes, in a study by Lortz et al, in 2000 (106). They investigated insulin-producing RINm5F cells that were bioengineered . . . against cytokine-mediated toxicity (106). Lortz and colleagues found that Catalase, Glutathione Peroxidase and Cu/Zn SOD were protective apparently because of an inactivation of ROS generated in the signal cascades of the cytokines used in the experiments (106).

Possel and co workers, (107), in 2002, investigated the protective effect of glutathione in preventing peroxynitrite formation from NO and .O₂ ⁻, generated simultaneously in microglia via iNOS, and NADPH-oxidase, respectively. Peroxynitrite formation in continuously nitric oxide-producing microglial cells was rather limited. However, activation of the superoxide-generating enzyme, NADPH-oxidase, dramatically increased (peroxynitrite) within a few minutes. We conclude that superoxide is the limiting factor for peroxynitrite formation”. Glutathione, but not ascorbate, significantly decreased peroxynitrite formation (107).

b. GSH Protects Mitochondria Vs. H₂O₂, Bioenergetic Failure, and Exaggerated Apoptotic Processes that Add to Cumulative Tissue Toxicities.

This subsection has three parts: (i) Summary Introduction; (ii) Glutathione transporters on plasma membranes and mitochondria, and protection against H₂O₂; and (iii) Maintenance of Glutathione concentrations protects mitochondria vs. Bioenergetic failure and may curb some apoptotic processes.

(i) Summary Introduction

Robillard and Konings, in 1982, placed an early emphasis on the concept that the control of membrane transport systems in mitochondria rests with the thiol redox state (108). Their proposal has been supported and expanded since that time as described in (ii) and (iii) below. The three major redox-active factors within mitochondria, in summary, are the electron transporters of the reparatory chain, protein thiols, and matrix glutathione. These are inter-related by the availability of NADPH, a co factor for glutathione reductase, which is essential for the maintenance of the redox state of intra mitochondrial glutathione. Free sulfhydryl groups, as in reduced glutathione, are essential for the activity of all metabolite carriers in the inner mitochondrial membrane, otherwise, extensive thiol oxidation results in:

-   -   the inhibition of transport activity, and     -   the dissipation of the mitochondrial inner transmembrane         potential (delta psi [m]), an early, potentially reversible         event observed in mitochondrial related apoptotic processes         (109), bioenergetic failure, also an early event that is part of         the dissipation of delta psi (m) (110), and an increase in the         permeability of the mitochondrial membrane, a later event in         mitochondrial related apoptotic processes (111, 112).

(ii) Glutathione Transporters, on Plasma Membranes and Mitochondria, and Protection Vs. H₂O₂

Mitochondrial glutathione concentrations are higher that those in the cytosol, 10 mM compared to 7 mM, respectively (113). Since mitochondria can not synthesize glutathione, for lack of the two enzymes (gamma glutamylcysteine synthase and glutathione synthase), it is transported (115-119) against a concentration gradient into the mitochondrial matrix by a high-affinity component (Km, approximately 60 micro M; V max, approximately 0.5 n mol/min per mg of protein) . . . stimulated by ATP and a lower affinity component (115).

The need for glutathione in mitochondria is its catalytic activity, to reduce H₂O₂ produced by MnSOD acting on superoxide anion (120-123). Since there is no catalase produced in, or transported into mitochondria (121, 124), mitochondrial glutathione is indispensable. In the cytosol of all cells, plasma, and red blood cells, superoxide dismutase (in the form of CuZn SOD) functions in concert with catalase to prevent accumulations of H₂O₂. Without catalase, or the catalytic activity of glutathione, H₂O₂ would accumulate, eventually producing highly injurious hydroxyl radicals (.OH) (66, 74). The consequences can lead to bioenergetic failure of strategic cells and to exaggerated, apoptotic processes.

(iii) Maintenance of Glutathione Concentrations Protects Mitochondria Vs. Bioenergetic Failure and May Curb Some Apoptotic Processes.

Bioenergetic Failure and its relation to glutathione depletion, with the later development of apoptotic processes, was discussed early by Slater, Orrenius and colleagues from the Karolinska Institute in 1995 (125). They indicated that GSH depletion will lower a cell's capacity to buffer against endogenous oxidants, (and) it may set a time limit on continued mitochondrial function and thus indirectly on total ATP levels and membrane integrity (125).

Macho, Kroemer and co-workers (109), in 1997, studied thymocytes and noted disruption of the mitochondrial transmembrane potential (delta psi [m]), and a depletion of non oxidized glutathione that was near-to-simultaneous (109). They felt this represented step 1 in a two step apoptotic process (109). The loss of delta psi (m) represents bioenergetic failure with diminished ATP synthesis. This state can progress to later apoptotic processes as step 2 (109).

Apoptotic processes sometimes benefit the patient, as in the eradication of cancer cells, and perhaps microbe-laden cells, provided the microbes in the cell fragments that are phagocytosed by non-infected cells are not infectious or are rapidly rendered so.

In 2002, Humlova and colleagues (126) reported their laboratory findings on apoptotic processes in vaccinia virus-infected macrophages (murine line J774.G8). Among their apoptotic criteria were changes of mitochondrial membrane potential Their studies demonstrated that in vaccinia virus (VV) infection early gene expression seem(ed) to be required for induction of apoptosis while late gene expression was not (126). In summary, they found that induction of apoptosis by VV in macrophages . . . induces a decrease in mitochondrial membrane potential and is associated with altered levels of Bcl-x (L) . . . an anti-apoptotic member of the Bcl-2 family (126).

Zaucha and co-workers at the U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Md., in 2001 (127) studied the pathology of aerosolized lethal doses of monkey pox virus in cynomolgus monkeys. Although monkey pox is not readily transmitted among humans (3) it, along with variola, vaccinia and cow pox cause cutaneous lesions that share similarities. For this reason, the delineation of apoptotic processes in monkey pox by Zaucha and colleagues is described here since variola has not been studied in humans with the modern techniques that provide insights into the molecular cyto- and tissue pathology that can help guide therapeutic developments.

The lower airway epithelium served as the principal target (127), but the involvement of lymphoid tissues in the tonsils, and mandibular nodes suggested these lymphoid tissues were infected early. The pathology indicated widespread lymphatic dissemination of the virus through a monocytic cell-associated viremia . . . (and) the mononuclear phagocyte dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites (127).

The observations of the early involvement of lymph nodes in the head and neck, the role of the monocytic cell in the viremias, and the targeting of the mononuclear phagocyte/dendritic cell system are reminiscent of the variola descriptions by the Henderson group (3), along with the suggestion that these cell types provided the means of entry into other systemic sites Henderson (3) and the older literature (2) found that the lodging of circulating, variola-infected mononuclear leucocytes in dermal vessels constituted the dissemination system into the skin. Guarneri bodies (difficult-to-find epithelial intracytoplasmic inclusion bodies) were present in the epidermis in monkey pox and variola infections, providing further pathologic similarities.

Lesions of monkey pox infection, described by Zaucha et al, were found at all sites to be necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis (127). Based on pathologic, and virologic similarities (monkey pox, vaccinia, cow pox, and variola are members of the genus orthopox virus, and can infect humans and cause cutaneous lesions (3)), it would seem that smallpox lesions also develop, in part, by apoptotic processes.

The molecular pathologic processes believed to occur in smallpox take a toll on glutathione concentrations [GSH]: (a) induction of iNOS with increased NO production, (b) the generation of superoxide, (c) formation of peroxynitrite, (d) glutathione-consuming chain reactions of ROS and RNS, (e) TNFα related oxidative stress, the induction of Cox-2 with increased oxidation of arachidonic acid, (g) oxidative bursts from macrophages engaged in phagocytosis at sites of infection and inflammation, and related cascades.

With losses of intracellular glutathione, the types of apoptotic processes induced in smallpox infections would include those driven by glutathione losses and diminished redox in mitochondria and cytosol.

In a review article (128), dealing with T lymphocyte apoptosis, Penninger summarized the role of mitochondria, including the transmembrane potential (delta psi (m)), the mitochondrial permeability transition pores (PT), the disruption of outer mitochondrial membrane integrity leading to the release of cytochrome c and apoptosis inducing factors, and the temporal aspects of several modifying factors which included glutathione.

Jones, Orrenius, and co-workers (129) found a significant amount of pro-caspase-3 in mitochondria. Their studies in Jurkat cells indicate the pro-caspase-3 is in a pre-apoptotic complex, within mitochondria with “chaperone” proteins.

Hall (130) described the role of glutathione in the regulation of apoptosis and the control of redox changes associated with apoptosis. The function of GSH in regulating mitochondrial membrane permeability was also reviewed.

Celli and co workers (131) demonstrated that in their apoptosis studies, wherein glutathione was depleted with buthionine sulfoximine, the Bcl-2 protein, which is crucial in preventing, or slowing apoptosis, was degraded. The beneficial effect of adding glutathione to cells in this study was not a direct effect of glutathione but possibly a prevention of the degradation of Bcl-2 protein (131).

Watson and co-workers (132) demonstrated that when Fas triggers apoptosis in human activated neutrophils, inhibition of the subsequent apoptotic processes may be achieved by increasing the intracellular glutathione levels with exogenous glutathione. They indicated that Fas induced signaling for neutrophil apoptosis can be inhibited in a redox sensitive manner (132).

Marchetti, with Kroemer and colleagues (133), in early work, studied the impact of thiol oxidation and redox on the induction of apoptotic processes. Their findings included that monovalent thiol-reactive compounds inhibit(ed) apoptosis induced by their model systems in thymocytes, and further, that the critical thiols . . . are likely to be located in the mitochondrial matrix (133).

Hirsch, with Kroemer and co-workers (134), as part of their inquiry into the factors involved in cell death by apoptosis, or by necrosis, also found that hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione were integral to their apoptotic models (134).

The presence of glutathione transporters on plasma membranes and on mitochondria has been well delineated and these provide the molecular/cellular means to assure intracellular and intra mitochondrial distribution of administered glutathione (114-119, 135).

c. Arachidonate Oxidation and GSH

(i) Summary Introduction

Glutathione (GSH), via its GSH peroxidases and GSH S-transferases, (81,82), helps control oxidations of arachidonate, thereby decreasing lipid hydroperoxides, isoeicosanoids and eicosanoids that can be overly proinflammatory and disadvantageously apoptogenic and immunosuppressive. Regarding terminology, eicosanoids include prostaglandins, thromboxanes, leukotrienes and lipoxins formed by enzymes, whereas isoeicosanoids are non enzymatically formed isomers driven by free radical reactions.

The GSH peroxidases and S-transferases are protective and normally disassemble lipid hydroperoxides before they can expand cell injury (81,82). But, they require glutathione which may become compromised by oxidative stress induced by several factors in the course of serious infections, such as oxidative bursts by activated macrophages, hypergeneration of superoxide, TNFα-generated oxidative stress, induction of Cox-2 with increased arachidonate oxidations, and induction of iNOS with increased formation of nitric oxides and nitrosated glutathione (15, 48, 66-75, 81, 82, 90-95, 113, 120, 121).

(ii) Brief Background of Peroxidation Chemistry

The C-2 of the glycerol backbone of phophoglycerides is generally esterified to a polyunsaturated fatty acyl chain such as arachidonate, and the C-2 ester may be acted on by phospholipase A2 (PLA₂) thereby releasing free arachidonate (69, 71). These polyunsaturates are highly subject to peroxidation, either spontaneously or enzymatically by cyclooxygenases and lipoxygenases, because their unsaturated (double bonded) carbons are un conjugated, i.e., separated by an interposed, singly bonded carbon, classified as alpha methylenic because its C—H bonds are activated due to the distribution of bonding electrons over a “spread” of four to five carbons (66, 69, 71). Abstraction of a hydrogen from the alpha methylenic carbon therefore occurs readily by molecular oxygen radicals, leaving a carbon radical that is instantly attacked by other activated species, with the final product generally being a hydroperoxide (—OOH) attached to the carbon (LOOH) (66, 69, 71).

These lipid peroxides are unstable and undergo schism, producing an alkoxy and a hydroxyl radical that can attack other alpha methylenic carbons: (66, 69, 71) [LOOH→LO.+.OH]

Peroxidation of arachidonate and other polyunsaturates can occur within membranes, and micelles, and their schism products can initiate cytotoxic chain reactions that alter membrane structure and also attack membrane proteins, and membrane associated DNA (66, 69-72).

There are a number of controls that protect cells from rampant lipid peroxidation, including inherent membrane structure, normally low levels of PLA₂ activity, lipophilic antioxidants, and enzymes that disassemble LOOH's before they can undergo schism, such as the glutathione peroxidases, and glutathione S-transferases (69-72, 82).

(iii) Relevance in Viral Infections

Vaccinia viruses (VV) that have been bioengeneered to express IL-4, as in (a) the 1996 work of Sharma et al (14), (b) the 1992 studies of Andrew and Coupar (24), and (c) the 1999 work of Aung and colleagues (25) in constructing recombinant VV to express a Respiratory Syncytial Virus epitope (M2), either alone or in combination with IL-4 (vv M2/IL-4), become more difficult to clear, fail profoundly (14) to elicit antiviral cytotoxic lymphocyte responses (14), and kill the infected laboratory animal more rapidly.

These disadvantageous effects of over expressed IL-4 in acute viral infections can occur without bioengineering. Insufficient intracellular glutathione concentrations resulting from aging (26, 46), diabetes (27, 28), prior exposures to chemical toxins (29, 31, 52), or during the course of serious infections (15-17, 21, 53), as outlined previously, will skew immune response patterns to Th2 pathways (13, 18-23) and result in disadvantageous up-regulation and increased expression of IL-4.

Among the IL-4 disadvantages during an acute viral infection, described under Property One and Property Two, is the up-regulation of the cyclooxygenases (COX-2), and the lipoxygenases (5-, 12-, 15-), resulting in excess formation of lipid hydroperoxides (LOOH's) and the simultaneous down-regulation of the protective enzymes that disassemble LOOH's (GSH peroxidases and GSH S-transferases) (82). Schnurr and colleagues (82) in 1999 included human peripheral monocytes in their studies of this inverse regulation by IL-4. Further, To find out whether these regulatory processes also occur in vivo, arachidonic acid oxygenase and phospholipid hydroperoxide glutathione peroxidase activity was assayed in various tissues of transgenic mice that systemically overexpress interleukin 4. In lung, spleen, kidney and heart, an increased arachidonic acid oxygenase activity was detected when transgenic mice were compared with inbred controls. The phospholipid hydroperoxide glutathione peroxidase activity was impaired in lung, liver, and spleen of the transgenic animals (82).

Sandstrom and co-workers (81) demonstrated 15-HPETE, derived from 15-lipoxygenase activity, and several other hydroperoxy fatty acids were lethal to a T cell line that is chronically HIV infected (8E5) because of a “marked reduction in glutathione peroxidase activity inherent in the 8E5 T cell line (81).

IL-4, therefore, when overexpressed by viral engineering, or insufficient glutathione concentrations that skew T helper response patterns to Th2, can result in lethal accumulations of hydroperoxyls by up-regulating 12- and 15-lipoxygenases to produce increased quantities of 12- and 15-HPETE's. These can not be reduced to the less toxic 12- and 15-HETE's because the relevant GSH peroxidases have been down-regulated by IL-4 (82).

In addition, in the course of acute inflammation and NFκB activation, Cox-2 is induced (47). Kim et al (47) demonstrated, however, that the induction of Cox-2 could be inhibited, in their model systems, by glutathione, N-acetyl cysteine, and pyrrolidine dithiocarbamate (PDTC) (47).

Glutathione therapy that effectively replenishes and maintains intracellular concentrations should be able to down regulate IL-4 produced as a result of overly favored Th2 response patterns (13, 18-23), and also correct the GSH insufficiencies that may lead to Th2 cytokines in people over 45 (26), in diabetics (27, 28), in the chemically exposed (29, 31, 52), and in those with serious, overwhelming infections (15-17, 21, 53). The results of GSH therapy could include down regulation of 12- and 15-lipoxygenases, up-regulation of GSH peroxidases, and down regulation of the NFκB cascades that include the induction of Cox-2. These favorable biochemical results would help lead to the prevention and reduction of cyto- and tissue toxicities associated with smallpox infections.

Th2 cytokines pose an additional disadvantage during an overwhelming infection in fostering expression of a group of phospholipase A2's (PLA 2) that are preferentially expressed in type 2 helper T cells (136). PLA2's release arachidonate by carrying out interfacial catalytic activity at the interface of the aqueous phases on either side of a cell membrane, and the condensed lipid phase that is the membrane. PLA2 enzymes are structurally diverse in mammalian cells and are responsive to different conditions. One group of PLA2's was found early on, in 1993, to be inducible by cytokines such as IL-1, TNFα and IL-6 and to be suppressed by anti-inflammatory glucocorticoids (137).

Ho and colleagues at the Brigham, in 2001 (136), identified a new group of PLA2's (group XII, specifically, GXII-2 PLA2) that “ . . . were selectively expressed in murine type 2 helper T (Th2) clones and in vitro differentiated mouse CD4 Th2 cells as compared with type 1 helper T clones and cells (136). Anti-CD3 stimulation up-regulated GXII PLA2's in Th2 cells compared to type 1 helper cells. This novel PLA2 group would enhance the responsiveness of Th2 cells through release of immediate second signals and generation of downstream eicosanoids (136). If Th2 response patterns are up-regulated during a serious infection because glutathione concentrations have been eroded by a multiplicity of factors, as described previously, the enhanced catalytic activity of a specific PLA2 in type 2 helper T cells will speed the cumulative cyto- and tissue toxicities.

Finally, lipid hydroperoxides (LOOH's) that are not reduced to their corresponding LOH's generate toxic, apoptogenic products, including 4-hydroxy nonenal (HNE) (138). Compton et al in 1998 (138) demonstrated that the LOOH product, HNE, induced significant tissue injury and apoptosis. Of therapeutic interest is the finding of Hardwick and co workers at Cambridge, UK, in 1999 (139), that glutathione, in their particular experimental model of monocyte-macrophages, protected against macrophage death by conjugation of HNE, thereby rendering it non-toxic . . . the last example of this property of glutathione of neutralizing reactive intermediates that otherwise cause cellular and tissue toxicities (139).

This closes the purposely truncated discussion of property number three of glutathione. It can prevent and reduce cyto- and tissue toxicities produced by multiple pathways which, although different, share a capacity for amelioration by glutathione by virtue of the carefully positioned thiol of GSH with a zwitterion from the gamma glutamyl residue.

4. As occurs with glutathione vs. HIV, the thiol moiety of glutathione is capable of safe, post translational protein modifications that may disable thermodynamically less stable viral proteins synthesized during infections. Variola proteins that may be hindered include those involved in viral replication, specifically, encoded viral DNA topoisomerases, and evasion of host immunologic defenses.

a. Introduction

Cysteine residues and amino acid sequences are among the major factors determining the folded conformation of proteins for structural and functional utility. Considerable “effort” is expended by the normal cell as it forms disulfides for intramolecular and interchain bonds that have the requisite thermodynamic stability for the particular protein. Some protein disulfides must be readily accessible in order to undergo the redox changes that allow for regulation of enzymes, orderly signaling and “responsive” receptors. Still other proteins are designed for rapid “turnover” and must be easily unfolded and dismantled in preparation for replacement.

The folding of normal host proteins is guided by complex, controlled interactions that stabilize the final folded state (140). A number of factors, in addition to the amino acid sequence, affect folding, including [H+] and enzymes such as protein disulfide isomerase (PDI), peptidyl propyl isomerases (PPIases), and molecular chaperones. PDI accelerates disulfide interchange in kinetically trapped intermediates, shuffling these disulfides until the most stable protein configuration is achieved (141). PDI speeds this shuffling by several thousand fold. PPIases speed cis to trans isomerization since the peptide bonds are almost always in the trans configuration (142). Molecular chaperones inhibit improper intermolecular interactions of protein intermediates, literally consuming ATP to untangle trapped intermediates (143). These steps are necessary for protein stability, and require considerable energy resources along with PDI's, PPI's, and molecular chaperones.

Barbirz and colleagues in Konstanz, Germany, in 2000 (144) studied a very potent molecular chaperone which is itself dependent on disulfide bonds (144). Hsp 33 (heat shock protein) has six cysteinyl residues. When these are in their chemically reduced form, Hsp33 is inactive and displays no folding helper activity (144). However, under oxidizing circumstances, such as hydrogen peroxide, Hsp 33 becomes active and resumes its functions in untangling trapped protein/polypeptide intermediates within the easily clogged protein-folding subcellular compartment of the endoplasmic reticulum. Mass spectrometry (matrix-assisted laser desorption/ionization MS) demonstrated that Hsp 33 activation by H₂O₂ was accompanied by the formation of two intramolecular disulfide bonds with Hsp33: Cys (232)-S—S Cys (234) and Cys (265)-S—S (268) Accessibility of the cysteinyl residues was also emphasized.

For example, Cys (239), one of the six cysteinyl residues, is poorly accessible when Hsp 33 is active, and remains reduced despite H₂O₂ exposure, and does not therefore react with the sixth cysteinyl, Cys (141), which is highly reactive toward chemical modifications, demonstrating that other factors such as amino acid sequence, and other amino acid/peptidyl properties also are important in protein folding.

In non malignant, non infected eukaryotic cells, considerable ATP-consuming “quality control” goes into disulfides, as previously summarized (140-143). Pox virus protein synthesis, by contrast, proceeds within cytoplasmic enclaves inside infected cells, and does not have the benefits of orderly testing of the thermodynamic stability of their protein disulfides, nor is there a reliable, sufficient supply of ATP to support the ongoing synthesis of the three groups of “testing” proteins (protein disulfide isomerases, peptidyl prolyl isomerases and molecular chaperones) (140-143). This provides a basis for the post translational selectivity of glutathione in safely reducing and/or glutathionylating the critical protein thiols of variola, without cytotoxicity, particularly DNA topoisomerase and the evasion proteins.

b. Apparently Non Cytotoxic, Post Translational Modifications of Critical HIV Proteins: Tat, Gp120, Protease

As cited in prior subsections, the redox environment within some viral infected host cells, such as mononuclear cells/macrophages, is generally low, secondary to the over-utilization of glutathione in curbing reactive intermediates generated by: TNFα; NADPH oxidase; the alkoxy and hydroxyl free radicals resulting from the homolytic schism of lipid hydroperoxides (LOOH's →LO.+.OH) produced in excess by the induced COX-2; and by the Reactive Nitrogen Species (RNS) that accompany iNOS induction.

Low glutathione concentrations in HIV and other infections (15, 16, 17) foster an oxidative environment conducive to disulfide formation and extensive protein folding in the viral proteins synthesized within the infected mononuclear cells/macrophages. HIV replication rates are rapid when glutathione redox decreases, triggering the redox-sensitive activation of NFκB, which in turn activates nuclear-integrated HIV proviral DNA through NFκB binding sites on the long terminal repeat (LTR) (15-17, 77-80). The HIV proteins would be expected to have their cysteinyl residues bound as disulfides in this oxidative environment.

Koken and co-workers, in 1994 (145), analyzed an encoded HIV Tat protein that specifically activated transcription from the viral long terminal repeat. They discovered that Tat activity is dramatically inhibited by the preincubation of the protein with strongly reducing agents. These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds (145).

Palamara and colleagues, in 1996 (146), found that exogenous glutathione (GSH) strongly suppresses . . . virus infectivity . . . budding and release of virus particles from chronically infected cells (either macrophages or lymphocytes), together with a selective decrease in the expression of gp120, the major envelope glycoprotein, rich in intra chain disulfide bonds, and thus potentially sensitive to the effect of a reducing agent such as GSH . . . GSH can interfere with late stages of virus replication similar to cells exposed to herpes virus type 1 (a DNA virus) or to Sendai (an RNA virus), showing that the suppression of virus replication by GSH is related to the selective inhibition of envelope glycoproteins (146).

Garaci and co workers, in 1997 (147), studied intracellular GSH content and HIV replication in human macrophages. In vitro HIV-1 infection induced a significant decrease in intracellular reduced glutathione (GSH) in human macrophages. Such a decrease was observed at the time of infection corresponding to maximum release of virus from infected cells and was not related to cell cytotoxicity . . . . Treatment of macrophages with BSO (buthionine sulfoximine) significantly increased the HIV yield in the supernatant. Exogenous GSH strongly suppressed the production of p24 gag protein as well as the virus infectivity . . . GSH antiviral effect occurred at late stages of virus replication and was related to the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds (147).

In addition to the disulfide research in HIV Tat protein, and gp120, Davis et al, in 1997 (148) examined the disulfides in HIV protease and the effects of glutathionylation.

Previous studies suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity . . . We examined diglutathionylated wild type protease (Cys-67-SSG, Cys-95-SSG) and the monogluthionylated protease mutants (C67A, Cys-95-SSG and C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin) . . . At low thioltransferase concentrations (5 nM), deglutathionylation occurred almost exclusively at Cys-95-SSG. With substantially more thioltransferase (100 nM) Cys-67-SSG was partially deglutathionylated but only at 20% of the rate of Cys-95-SSG reduction. Treatment of the diglutathionylated protease with thioltransferase not only restored protease activity but generated an enzyme preparation that had a 3- to 5-fold greater specific activity relative to the fully reduced form . . . . Our results implicate thioltransferase in the regulation and/or maintenance (148).

Shatrov and associates, in 1996 (83), demonstrated that gp120 protein of HIV not only serves as the means of attachment of the virus to CD4+ receptors, it also amplifies the activity of a toxic cytokine, TNFα (causes free radical reactions and also weight loss). TNFα also activates NFκB, which in turn stimulates HIV replication. Hence, gp120 protein potentiates TNFα, which activates NFκB, and increases HIV replication. The gp120 protein alters the redox state of cells, and increases H₂O₂ production, ensuring disulfide formation in proteins.

This activity of gp120 causes a reduction of GSH and an increase in oxidized GSSG. The gp120 protein exerts these effects through the p56 1ck protein tyrosine kinase, which transmits a signal that increases free radical oxidative stress (83).

In these HIV studies (83, 145-148), wherein critical HIV disulfides were biochemically reduced, to the detriment of HIV, there were no findings apparently of cytotoxicity. In other HIV studies (80), where exogenous glutathione was added, and viral inhibition was evident, specific observations were made regarding the sustained anti HIV effects of glutathione for 35 days, in vitro, without producing detectable cellular toxicity in monocytes/macrophages . . . . ”

c. Viral DNA Topoisomerases

(i) Introduction

Topoisomerases are sulfhydryl proteins that nick one or both strands of supercoiled, double stranded DNA for processes that require separating the strands, for example, transcription, recombination and replication. This is accomplished by cleaving the DNA, one or both strands; and religation of the DNA break(s). There are many topoisomerases, and James Wang, one of the discoverers of these enzymes, has described them further (149). The two major categories, among others, are Type I topoisomerases that cleave only one strand, and Type II, which cleave both strands.

After Type II topoisomerases cleave the DNA (requires ATP), the 5′-phosphate terminus of each cleaved DNA strand remains attached to the enzyme by linking to a specific tyrosine residue at the active site (149). The two ends of the cleaved DNA, therefore, remain “anchored” to the topoisomerase. Otherwise, the two cleaved ends will rotate freely and massively disturb the topology and functions of the DNA.

Topoisomerases have been successfully targeted for antibiotics vs. bacteria, and chemotherapeutic agents vs. cancer (150). With regard to antibiotic use, DNA gyrase (150) in prokaryotes is more sensitive than the one in eukaryotes. The quinolone antibiotics are powerful pharmaceuticals and specifically inhibit topoisomerase II (DNA gyrase) and topoisomerase IV. The concept of differential sensitivities of topoisomerases to inhibitors in viruses, compared to human cells is relevant. A particular vulnerability of all topoisomerases is their complex mechanisms of action in modifying the topology of DNA by generating transient double-strand breaks. The dual function of Topoisomerases, i.e., catalysis and religation, requires unimpeded reactivities of the cysteinyl residues that bind and affect the inter-subunit interactions of topoisomerase to achieve “global” effects.

These cysteinyl residues are accessible and have been targeted. Quinoid inhibitors are described below. In addition to the reactivity and accessibility of these protein thiols, the active site Tyr-274 can serve as a target for nitric oxide which is generated by the viral-laden, activated macrophages. The formation of Nitrotyrosine, within the infected, nitric oxide-generating macrophages, may be enhanced by glutathione actions: (a) reducing the protein disulfides, and/or glutathionylating the free protein thiols allows for some unfolding, as intramolecular and interchain disulfides are reduced and/or glutathionylated; the subunits of topoisomerases rely, in part on inter chain disulfides for their dual actions of catalysis and religation; and (b) preventing peroxynitrite formation and thereby maintaining nitric oxide availability for nitrosation of Tyr-274; Nitrosoglutathione forms spontaneously and serves as an “orderly” nitric oxide donor.

(ii) Topoisomerase Inhibition in Pharmacology

Wittschieben et al, in 1998 (151) demonstrated that replacement of the active site tyrosine of vaccinia DNA topoisomerase converts the enzyme into a site-specific endonuclease Normally, Tyr-274 of this enzyme links to a specific phosphate in the DNA via a 3′-phosphodiester bond (vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5′-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3′-phosphodiester bond to Tyr-274 of the enzyme . . . . ”). Substitutions of Tyr-274 by glutamate, cysteine or histidine remain enzymatically active insofar as cleaving the duplex DNA, but religation does not occur since the 3′-phosphodiester bond doesn't form . . . it requires the OH of Tyr-274.

The product of the mutant substitutions is an endonucleolytically cleaved 60 bp duplex DNA at the CCCTT downward arrow site with a 3′-phosphate termination (151).

With biochemical reduction of the viral protein cysteinyl residues, or their glutathionylation, the active site tyrosine, Try-274, may become exposed and available for nitrosating reactions, to form Nitrotyrosine (99,101). Glutathione decreases aberrant reactions of Reactive Nitrogen Species (RNS), and protects Nitric Oxide so that peroxynitrite is less likely to form, and more is available for antiviral protein nitrosation (103-107).

Since the virus is within infected, activated mononuclear cells/macrophages, which now generate increased Nitric Oxide, secondary to the induction of iNOS, there would be a measure of selectivity in forming Nitrotyrosine in viral topoisomerase within these cells and thereby safely inhibit the enzyme.

Liu, a leader in topoisomerase research, investigated whether thiol alkylation of topoisomerase II could interrupt “global” activity of this enzyme. Liu and colleagues, in 2001, (152) employed a variety of thiol-reactive compounds, including quinones that react with thiols, and three other potent, thiol-reactive agents: N-ethyl maleimide (NEM); disulfiram; and an organic disulfide [2,2′ dithiobis (5-nitropyridine)]. All of these caused topoisomerase II-mediated DNA cleavage. The topoisomerase acted as an endonuclease (152). Liu and colleagues offered confirmation that these agents in fact reacted adversely with cysteinyl residues in topoisomerase by using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2) (152). This replacement “completely abolished” the TOP2-mediated DNA cleavage induced by thiol-reactive quinones (152). This work demonstrates the accessibility of topoisomerase cysteinyl residues, in some cases, to thiolating substances, e.g. glutathione, that can glutathionylate accessible cysteines, as shown in the prior subsection on HIV.

Neder and colleagues, at Wisconsin, in 1998 (153), demonstrated that the thiol residues on the topoisomerase II-DNA complex were exposed and were alkylated by naphthoquinones, thereby providing a biomimetic model of topoisomerase II poisoning by quinones (153).

Srinivasan and co workers, in 2002 (154), further demonstrated the accessibility and reactivity of protein sulfhydryl groups in topoisomerase II. They used this enzyme as a model system of a sulfhydryl-containing enzyme to determine the reactivities of the quinone metabolites of chlorinated biphenyls (environmental contaminants). They showed that the binding of these quinones to glutathione paralleled their binding to protein sulfhydryl groups in topoisomerase II, and offered this as the mechanism for the observed DNA strand breaks generated by PCB metabolism. This study adds further to the view that topoisomerase cysteinyl residues may be accessible and susceptible to exogenous glutathione, with perhaps greater vulnerability by viral topoisomerases, analogous to some bacterial topoisomerase inhibitors, like quinolone antibiotics vs. the topoisomease II (DNA gyrase) of a number of bacteria (150).

d. Evasion of Host Immunologic Defenses

(i) Introduction

The formation of intramolecular and interchain disulfide bonds, to fold proteins into their optimum configurations, proceeds under normal circumstances through “quality control” testing, as previously described [PDI's, PPIases, molecular chaperones, abundant ATP for the chaperones and for the syntheses of these “quality control” proteins, (140-143)].

By contrast, viral proteins, ex HIV Tat, gp120, and protease, (145-148), are generally produced under less than optimum circumstances because the intracellular environment of a viral infected cell, that is activated and responding, is chaotic, with compromised bioenergetics and host responses that include nitric oxide from NFκB induced iNOS (15-17, 47-51, 54-56, 77, 81-83, 98). The nitric oxide, unless diverted to form peroxynitrite, is available to form adducts with tyrosyl residues and cysteinyl residues that are relatively more exposed when the viral proteins are forming as nascent polypeptide chains. The potential to form Nitrotyrosine and Nitrosothiols in these nascent chains, within activated, infected mononuclear cells/macrophages may be significant (99-107).

However, the pathologically successful pox viruses, such as smallpox, encode proteins that allow the viruses to evade host immunologic responses (155-165). These evasion proteins, as described more fully below, correlate with virulence (155-165). They interact, directly and indirectly, with host defense factors to nullify them: NFκB, TNFα, IFN γ, IL-18, most of the components of the proinflammatory cytokine cascades, complement, chemokines, and inhibitors of dendritic cells, and thereby negate innate immunity via NK cells, and acquired immunity through T cells.

The result in the lethal pox virus infections is early, “silent”, unimpeded viral replication, and spread. When host cells do begin to react by inflammatory responses, around 10 days post variola exposure, defense factors are overwhelmed by the large numbers of virions. The patient is now prostrate, with hyperpyrexia, and facing death (2,3).

An erosion of intracellular glutathione, which is most severe in the responding mononuclear cells/macrophages and T cells, shifts the Th0 cells into Th2 response patterns that include increased IL-4 production, with suppression of: IL-12 IFN γ, and specific anti-variola cytolytic cells, the opposite of what is required to have a chance at recovery.

Based on the properties of glutathione, the nature of preferred glutathione formulation, and the ubiquitous presence of glutathione transporters, the following reactions should occur with glutathione administration:

Accessible cysteinyl residues in pox proteins (for virion structure, for viral replication, and for evasion) will be reduced, if present as thermodynamically inadequate disulfides; and

Exposed cysteinyl residues, particularly on the nascent variola polypeptide chains, can be glutathionylated through formation of mixed disulfide bonds.

(ii) The Host Evasion Proteins

Buller and Palumbo, from the National Institute of Allergy and Infectious Diseases, in 1991 (155), reviewed poxvirus pathogenesis up to that point, citing the unique features of this highly successful family of pathogens (155). Perhaps most surprising of all, the genomes of these viruses encode many proteins which interact with host processes at both cellular and systemic levels some of which blocked the development of a chemotactic substance, or interfered with the activation of the classical complement pathway. Their earlier work in 1989 (156) discovered a 38-kDa protein of cowpox virus that inhibited the generation of chemotactic molecules which are elicited during virus replication (156).

In 1993, Massung and a group under the Centers for Disease Control and Prevention (157) published their work wherein they determined the entire nucleotide sequence of the highly virulent variola major virus, strain Bangladesh-1975 (VAR-BSH; 186, 102 base pairs, 33.7% G+C; Genbank accession number, L22579) of the 187 proteins thought to be involved in pathogenicity and host-range, 150 were markedly similar to those of vaccinia virus . . . the remaining 37 proteins reflected variola-specific sequences (157). This was followed up with their publication in 1994 (158) wherein the proteins that were novel to variola were thought to possibly augment variola virus transmissibility and virulence for its only natural host, humans (158).

This area of viral research was reviewed by Alcami, and Smith (GL) in 1997 (159). They provided examples of evasion proteins vs. host responses: oppose apoptosis, capture chemokines, counteract complement, interfere with interferon, and intercept interleukins (159). Their later work and that of others, published through 2002, covered a number of factors in this arena of “stealth viruses” (160-165)

The following represent a sampling of the range of evasion proteins encoded by pox viruses and produced by infected cells: IL-18 binding proteins by ectromelia, vaccinia and cow pox, V. P. Smith, N. A. Bryant and A. Alcami, in 2000 (160); TNF receptors by cowpox, ectromelia, and camelpox, M. Saraiva, and A. Alcami, in 2001 (161); CC-chemokine inhibitor by vaccinia virus 35-kDa protein (VV-35 kDa), Seet and colleagues, 2001, (162); NM inhibition by cowpox virus, Oie and Pickup, 2001 (163); Complement regulatory protein by vaccinia virus, inactivates human C3b and C6; Rosengard and coworkers, 2002 (164); CD 30 homologue by ectromelia, induces reverse signaling in cells, blocks the generation of interferon gamma-producing cells, and strongly inhibits Th1 responses, but not Th2, M. Saraiva . . . A. Alcami, 2002, (165).

High GSH levels have been demonstrated to be necessary for proper functioning of platelets, vascular endothelial cells, macrophages, cytotoxic T-lymphocytes, and other immune system components. Recently it has been discovered that HIV-infected patients exhibit low GSH levels in plasma, in other fluids, and in certain cell types like macrophages, which does not appear to be due to defects in GSH synthesis. See: Droge, W., Pottmeyer-Gerber, C., Schmidt, H. & Nick, S.: Glutathione augments the activation of cytotoxic T lymphocytes in vivo. Immunobiol. 172:151-156, 1986.; Droge, W., Eck, H. P., Gmunder, H., and Mihm, S.: Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives. Amer. J. Medicine 91(3C):140S-144S, 1991.; Furukawa, T., Meydani, S. N. & Blumberg, J. B.: Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech. Ageing Dev. 38:107-117, 1987.; Franklin, R. A., Yong, M. L., Arkins, S., and Kelley, K. W.: Glutathione augments in vitro proliferative responses of lymphocytes to concanavalin A to a greater degree in old than in young rats. J. Nutr. 120:1710-17, 1990.; Kavanaugh, T. J., Grossman, A., Jaecks, E. P, Jinneman, J. C., Eaton, D. L., Masrtin, G. M., and Rabinovitch, P. S.: Proliferative capacity of human peripheral lymphocytes sorted on the basis of glutathione content. J. Cell. Physiol. 145:472-80, 1990.; Robinson, M. K, Rodrick, M. L., Jacobs, D. O., Rounds, J. D., Collins, K. H., Saproschetz, I. B., Mannick, J. A., and Wilmore, D. W.: Glutathione depletion in rats impairs T-cell and macrophage immune function. Arch. Surg. 128:29-35, 1993.; Suthanthiran, M., Anderson, M. E., Sharma, V. K. & Meister, A.: Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. Natl. Acad. Sci. USA 87:3343-3347, 1990.

I. Use of GSH to Treat AIDS

GSH has been shown to inhibit HIV replication in chronically-infected cells and in cells acutely infected in vitro. This makes GSH replacement therapy attractive, because it has the potential to interfere with the expression of the integrated HIV genome, a site that is not attacked by the currently employed antiretrovirals (AZT, ddI, ddC, D4T). GSH may also have benefits in countering the excess free radical reactions in HIV infection, which may be attributable to: 1) the hypersecretion of TNF-α by B-lymphocytes, in HIV infection, and 2) the catalysis of arachidonic acid metabolism by the GP-120 protein of HIV. The physiologic requirements for GSH by key cell types of the immune system, and the ability of macrophages to take up intercellular GSH, as well as to metabolically interact with T-lymphocytes to indirectly cause their GSH to increase, offer additional reasons to attempt to correct the GSH deficiency in HIV/AIDS.

In other new data dealing with HIV infections, the March 1997 issue of the Proceedings of the National Academy of Sciences (PNAS) established GSH deficiency as a key determinant of survival in HIV disease GSH deficiency is associated with impaired survival in HIV disease (PNAS. Vol. 94, pp. 1967-1972). The quest to raise GSH levels in cells is widely recognized as being extremely important in HIV/AIDS and other disorders, because the low cellular GSH levels in these disease processes permit more and more free radical reactions to propel the disorders.

HIV is known to start pathologic free radical reactions that lead to the destruction of GSH, as well as exhaustion of other antioxidant systems and destruction of cellular organelles and macromolecules. In pre-clinical studies, GSH stops the replication of the virus at a unique point, and specifically prevents the production of toxic free radicals, prostaglandins, TNF-α, interleukins, and a spectrum of oxidized lipids and proteins that are immunosuppressive, cause muscle wasting and neurological symptoms. Restoring GSH levels could slow or stop the diseases progression, safely and economically.

In mammalian cells, oxidative stresses, i.e., low intracellular levels of reduced GSH, and relatively high levels of free radicals, activate certain cytokines, including NFκB and TNF-α, which, in turn, activate cellular transcription of the DNA to mRNA, resulting in translation of the mRNA To A Polypeptide Sequence. See, Sonia Schoonbroodt, Sylvie Legrand-Poels, Martin Best-Belpomme and Jacques Piette; Activation of the NF-κB transcription factor in a T-lymphocytic cell line by hypochlorous acid, Biochem. J. (1997) 321, 777-785, Flohé, L., Brigelius-Flohé, R., Saliou, C., Traber, M. G. and Packer, L., Redox regulation of NF-κB activation. (1997) Free Radical Biology and Medicine, 22:1115-1126. Antioxidants have been shown to block the induction of NFκB by oxidant agents. In a virus-infected cell, the viral genome is transcribed, resulting in viral RNA production, generally necessary for viral replication of RNA viruses and retroviruses. These processes require a relatively oxidized state of the cell, a condition which results from stress, low glutathione levels, or the production of reduced cellular products. The mechanism that activates cellular transcription is evolutionarily highly conserved, and therefore it is unlikely that a set of mutations would escape this process, or that an organism in which mutated enzyme and receptor gene products in this pathway would be well adapted for survival. Thus, by maintaining a relatively reduced state of the cell (relatively reduced redox potential), viral transcription, a necessary step in late stage viral replication, is impeded.

The amplification effect of oxidative intracellular conditions on viral replication is compounded by the actions of various viruses and viral products that degrade GSH. For example, GP-120, an HIV surface glycoprotein having a large number of disulfide bonds, and normally present on the surface of infected cells, oxidizes GSH, resulting in reduced intracellular GSH levels. On the other hand, GSH reduces disulfide bonds of GP-120, decreasing or eliminating its biological activity, which in turn is necessary for viral infectivity. GSH therefore interferes with the production of such oxidized proteins, and degrades them once formed. GSH also participates in the destruction of hydrogen peroxide, which is a long-lived oxidative messenger which has been implicated in activating NM. R. Schreck, P. Rieber & P. A. Baeuerle; Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1, EMBO J 10: 2247-2258 (1991).

In a cell which is actively replicating viral gene products, a cascade of events may occur which allow the cell to pass from a relatively quiescent stage with low viral activity to an active stage with massive viral replication and cell death, accompanied by a change in cellular redox potential; by maintaining adequate GSH levels, this cascade may be impeded.

Thus, certain viral infections, such as HIV, are associated with reduced GSH levels, and it is believed that by increasing intracellular GSH levels in infected cells, as well as increasing extracellular GSH, the replication of HIV may be interfered with, and the cascade of events delayed or halted. It is noted that AIDS may also be associated with reduced GSSG levels, implying an interference with de novo synthesis of GSH as well as the oxidation of existing GSH discussed above.

Initially after infection with HIV, there is an intense viral infection simulating a severe case of the flu, with massive replication of the virus. This acute phase passes within weeks, spontaneously, as the body mounts a largely successful immune defense. Thereafter, the individual has no outward manifestations of the infection. However, the virus continues to replicate, insidiously, within immune system tissues and cells, like lymph nodes, lymphoid nodules and special multidendritic cells that are found in various body cavities.

This infection is not just a viral problem. The virus, in addition to replicating, causes excessive production of various free radicals and various cytokines in toxic or elevated levels. The latter are normally occurring biochemical substances that signal numerous reactions, usually existing in minuscule concentrations. Eventually, after an average of 7-10 years of seemingly quiescent HIV infection, the corrosive free radicals and the toxic levels of cytokines begin to cause symptoms, and failures in the immune system begin. Toxic factors, such as 15-HPETE, which is immunosuppressive, and TNF-α, which causes muscle wasting, are produced. The numbers of viral particles increase and the patient develops the Acquired Immune Deficiency Syndrome, AIDS, which may last 2 to 4 years before the individual's demise. AIDS, therefore, is not simply a virus infection, although the viral infection is believed to be an integral part of the etiology of the disease.

HIV has a powerful ability to mutate. It is this capability that makes it difficult to create a vaccine or to develop long-term anti-viral pharmaceutical treatments. As more people continue to fail the present complex pharmaceutical regimens, the number of resistant viral strains is increasing. This is a particularly dangerous pool of HIV and poses a considerable threat. These resistant mutants also add to the difficulties in developing vaccines. This epidemic infection is out of control, and the widely popularized polypharmaceutical regimens that are aimed only at lowering the number of viruses are proving to be too complex, too toxic, too costly, and too narrow. As a result, since the introduction of protease inhibitors, in combination with AZT-type drugs, increasing numbers of people are failing such therapies. Further, the continuing production of free radicals and cytokines, which may become largely independent of the virus, perpetuates the dysfunctions of the immune system, the gastrointestinal tract, the nervous system, and many other organs in AIDS. The published scientific literature indicates that many of these diverse organ system dysfunctions are due to systemic GSH deficiencies that are engendered by the virus and its free radicals. GSH is consumed in HIV infections because it is the principal, bulwark antioxidant versus free radicals. An additional cause of erosion of GSH levels is the presence of numerous disulfide bonds (—S—S—) in HIV proteins, such as the GP-120 discussed above. Disulfide bonds react with GSH and oxidize it.

The current HIV/AIDS pharmaceuticals take good advantage of the concept of pharmaceutical synergism, wherein two different targets in one process are hit simultaneously. The effect is more than additive. The drugs now in use were selected to inhibit two very different points in the long path of viral replication. The pathway of viral replication can be depicted simply:

HIV Replication Pathway point #1 point #2 point #3 point #4 point #5 Virus attacks and Virus makes DNA Viral DNA is Proviral DNA is inactive for a Viral RNA is produced, along enters the cell from its RNA integrated into long time, but activators will with viral membranes and Viral gp120 protein Reverse cells′ DNA start HIV replicating rapidly proteins, which are and CD4+ cell transcriptase is the Integrase is the NF κ B is the activator of assembled receptors and others enzyme involved enzyme involved dormant HIV DNA and Viral protease is involved are involved glutathione levels must be low for activation to occur AZT, ddl, ddC Glutathione Protease Inhibitors

Point #2 was the earliest point of attack, using AZT-types of drugs, including ddI, ddC and others. These are toxic and eventually viruses become resistant to these Reverse Transcriptase inhibitors.

Point #5 is a late replication step, and this is where protease inhibitors function. The drug blocks viral protease, an enzyme that snips long protein chains to just the right length so the viral coat fits exactly around the nucleic acid core, and that proteins having different biological activities are separated. By themselves, protease inhibitors foster the rapid development of resistant, mutant strains.

By combining Reverse Transcriptase inhibitors plus protease inhibitors, synergism was obtained and the amounts of viral particles in the plasma plummeted, while the speed of the developing mutant resistant viral strains was slowed, compared to using only one type of inhibitor. The initial promise of these combination therapies or “cocktails” has been tainted by increasing numbers of failures, which are expected to rise as resistant mutants develop, albeit more slowly than the use of the drugs separately.

New therapies include additional drugs in the classes of Reverse Transcriptase inhibitors and protease inhibitors. Also, drugs are in development to block point #3, wherein the enzyme, integrase, integrates the HIV DNA into the infected cell's DNA, analogous to splicing a small length of wire into a longer wire. Vaccine development also continues, although prospects seem poor because HIV appears to be a moving target and seems to change as rapidly as a chameleon. Vaccine development is also impaired by the immune cell affinity of the virus.

Human Immunodeficiency virus-infected individuals have lowered levels of serum acid-soluble thiols and GSH in plasma, peripheral blood monocytes, and lung epithelial lining fluid. In addition, it has been shown that CD4+ and CD8+ T cells with high intracellular GSH levels are selectively lost as HIV infection progresses. This deficiency may potentiate HIV replication and accelerate disease progression, especially in individuals with increased concentrations of inflammatory cytokines because such cytokines stimulate HIV replication more efficiently in GSH-depleted cells. GSH and glutathione precursors such as N-acetyl cysteine (NAC) can inhibit cytokine-stimulated HIV expression and replication in acutely infected cells, chronically infected cells, and in normal peripheral blood mononuclear cells.

It is noted that depletion of GSH is also associated with a processes known as apoptosis, or programmed cell death. Thus, intercellular processes that artificially deplete GSH may lead to cell death, even if the underlying process itself is not lethal. See: Arpadi, S. M., Zang, E, Muscat J. and Richie, J.: Glutathione deficiency in HIV-1-infected children with growth failure, (submitted for publication).; Baker, D. H. and Wood, R. J.: Cellular antioxidant status and human immunodeficiency virus replication. Nutr. Rev. 50:15-8, 1992.; Baruchel, S., and Wainberg, M. A.: The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus. J. Leukocyte Biol. 52:111-114, 1992.; Buhl, R., Holroyd, K. J., Mastrangli, A., Cantin, A. M., Jaffe, H. A., Wells, F. B., Saltini, C. and Crystal, R. G.: Systemic glutathione deficiency in symptom-free HIV-seropositive individuals. Lancet ii:1294-1298, 1989.; de Quay, B., Malinverni, R. and Lauterburg, B. H.: Glutathione depletion in HIV-infected patients: role of cysteine deficiency and effect of oral N-acetylcysteine. AIDS 6:815-9, 1992.; Droge, W., Eck, H. P. and Mihm, S.: HIV-induced cysteine deficiency and T-cell dysfunction—a rationale for treatment with N-acetylcysteine. Immunol. Today 13:211-4, 1992.; Eck, H. P., Gmunder, H., Hartmann, M., Petzoldt, D., Daniel, V.; Droge, W.: Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1-infected patients. Biol. Chem. Hoppe-Seyler 370: 101-108, 1989.; Fauci, A. S.: Multifactorial nature of human immunodeficiency virus disease: Implications for therapy. Science 262:1011-1018, 1993.; Foley, P. Kazazi, F., Biti, R., Sorrell, T. C., and Cunningham, A. L.: HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens via production of eicosanoids. Immunology 75:391-97, 1992.; Hasan, V., Thomas, D., Aclami, J. et al.: Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NM translocation but not human immunodeficiency virus 1 enhancer-dependent transcription. Proc. Natl. Acad. Sci. 87:7861-65, 1990.; Ho, W. Z. and Douglas, S. D.: Glutathione and N-acetylcysteine suppression of human immunodeficiency virus replication in human monocyte/macrophages in vitro. AIDS Res. Hum. Retroviruses, 8:1249-53, 1992.; Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L.: Redox status of cells influences constitutive or induced NF?B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149:3386-93, 1992.; Kobayashi, S., Hamamoto, Y., Kobayashi, N., and Yamamoto, N.: Serum level of TNFa in HIV-infected individuals. AIDS 4:169 1990.; Kalebic, T., Kinter, A., Poli, G., Anderson, M. E., Meister, A. and Fauci, A. S.: Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. Proc. Natl. Acad. Sci. USA 87: 986-990, 1991.; LeGrand-Poels, S., Vaira, D., Pincemail, J., Van de Vorst, A. and Piette, J.: Activation of human immunodeficiency virus type 1 by oxidative stress. AIDS Res. Hum. Retrov. 6:1389-97, 1990.; Mihm, S., Ennen, J., Pessara, U., Kurth, R.; Droge, W.: Inhibition of HIV-1 replication and NFκB activity by cysteine and cysteine derivatives. AIDS 5:497-503, 1991.; National Institutes of Health. Dr. Howard C. Greenspan, Chairman of Conference on Free Radicals and Antioxidants in HIV/AIDS, Nov. 12-13, 1993/Greenspan, H. C. The role of reactive oxygen species, antioxidants and phytopharmaceuticals in human immunodeficiency virus activity. Med-Hypotheses 40: 85-92, 1993.; Roederer, M., Raju, P. A., Staal, F. J. T., Herzenberg, L. A. and Herzenberg, L. A.: N-acetylcysteine inhibits latent HIV expression in chronically infected cells. AIDS Res. Human Retrovir. 7:(6) 563-567, 1991.; Roederer, M., Staal, F. J. T., Osada, H., Herzenberg, L. A. and Herzenberg, L. A.: CD4 and CD8 T cells with high intracellular glutathione levels are selectively lost as the HIV infection progresses. Internat. Immunol. 3:933-37, 1991.; Roederer, M., Staal, F. J. T., Raju, P. A., Ela, S. W., Herzenberg, L. A. and Herzenberg, L. A.: Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine. Proc. Natl. Acad. Sci. USA 87: 4884-4888, 1990.; Schreck, R. Rieber, P., and Baeurle, P. A.: Reactive oxygen intermediates as apparently widely used messengers in the activation of the NFκB transcription factor and HIV-1. EMBO J. 10:2247-2258, 1991.; Staal, F J., Roederer, M., Herzenberg, L. A. and Herzenberg, L. A.: Glutathione and immunophenotypes of T and B lymphocytes in HIV-infected individuals. Ann. NY Acad. Sci. 651:453-63, 1992.; Staal, F. J. T., Roederer, M. Herzenberg, L. A., and Herzenberg, L. A.: Intracellular thiols regulate activation of nuclear factor κ-B and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87: 9943-9947, 1990.; Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., Herzenberg, L. A. and Herzenberg, L. A.: Glutathione deficiency and human immunodeficiency virus infection. Lancet 339:909-12, 1992.; Staal, F. J., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A., McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A., Herzenberg, L. A. and Herzenberg, L. A.: Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals. AIDS Res. Hum. Retroviruses 8:305-11, 1992.; Staal, F. J. T., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W., Herzenberg, L. A., and Herzenberg, L. A.: Antioxidants inhibit stimulation of HIV transcription. AIDS Res. Hum. Retrov. 9:299-306, 1993.; Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Harel-Bellan, A. and Farrar, W. L.: Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1. Proc. Natl. Acad. Sci. 86:621-625, 1989.

II. Use of GSH to Treat Diabetes Mellitus

Diabetes mellitus is found in two forms, childhood or autoimmune (type I, IDDM) and late-onset or non-insulin dependent (type II, NIDDM). The former constitute about 30% and the remainder represent the bulk of cases seen. Onset is generally sudden for Type I, and insidious for Type II. Symptoms include excessive urination, hunger and thirst with a slow steady loss of weight in the first form. Obesity is often associated with the second form and has been thought to be a causal factor in susceptible individuals. Blood sugar is often high and there is frequent spilling of sugar in the urine. If the condition goes untreated, the victim may develop ketoacidosis with a foul-smelling breath similar to someone who has been drinking alcohol. The immediate medical complications of untreated diabetes can include nervous system symptoms, and even diabetic coma.

Because of the continuous and pernicious occurrence of hyperglucosemia (very high blood sugar levels), a non-enzymatic chemical reaction occurs called glycation. Since glycation occurs far more frequently inside cells, the inactivation of essential enzyme proteins happens almost continually. One of the most critical enzymes, γ-glutamyl-cysteine synthetase, is glycated and readily inactivated. This enzyme is the crucial step in the biosynthesis of glutathione in the liver.

The net result of this particular glycation is a deficiency in the production of GSH in diabetics. Normally, adults produce 8-10 grams every 24 hours, and it is rapidly oxidized by the cells. GSH is in high demand throughout the body for multiple, essential functions, for example, within all mitochondria, to produce chemical energy called ATP. Brain cells, heart cells, and others simply will not function well and can be destroyed through apoptosis.

GSH is the major antioxidant in the human body and the only one we are able to synthesize, de novo. It is also the most common small molecular weight thiol in both plants and animals. Without GSH, the immune system cannot function, and the central and peripheral nervous systems become aberrant and then cease to function. Because of the dependence on GSH as the carrier of nitric oxide, a vasodilator responsible for control of vascular tone, the cardiovascular system does not function well and eventually fails. Since all epithelial cells seem to require GSH, the intestinal lining cells don't function properly and valuable micronutrients are lost, nutrition is compromised, and microbes are given portals of entry to cause infections.

The use of GSH precursors cannot help to control the GSH deficiency due to the destruction of the rate-limiting enzyme by glycation. As GSH deficiency becomes more profound, the well-known sequellae of diabetes progress in severity. The complications described below are essentially due to runaway free radical damage since the available GSH supplies in diabetics are insufficient. See: Ceriello, A., Giugliano, D., Quatraro, A. & Lefebvre, PI: Anti-oxidants show an anti-hypertensive effect in diabetic and hypertensive subjects. Clin. Sci. 81:739-742, 1991.; Paolisso, G., Giugliano, D., Pizza, G., Gambardella, A., Tesauro, P., Varricchio, M. & D'Onofrio, F.: Glutathione infusion potentiates glucose-induced insulin secretion in aged patients with impaired glucose tolerance. Diabetes Care 15:1-7, 1992.

Reducing sugars are known to interact with free amino groups in proteins, lipids, and nucleic acids to form Amadori product and produce reactive oxygen species through the glycation reaction. Under diabetic conditions, glucose level is elevated and the glycated proteins increased. Cu,Zn-SOD has been shown to be glycated and inactivated under diabetic conditions and that ROS produced from the Amadori product caused site-specific fragmentation of Cu,Zn-SOD. Fructose, which is produced through polyol pathway, has stronger glycating capacity than glucose because the physiologic proportion of the linear form is higher than that of cyclized form. Fructose, as well as ribose, can bring about apoptosis in pancreatic β islet cell line. Levels of intracellular peroxides, protein carbonyls, and malondialdehyde are increased in the presence of fructose. In addition, methylglyoxal and 3-deoxyglucosone have also been shown to induce apoptotic cell death. 3-Deoxyglucosone, a 2-oxoaldehyde, is produced through the degradation of Amadori compounds. Both compounds are elevated during hyperglycemia and accelerate the glycation reaction. These compounds are toxic to cells, due to their high reactivity, and a scavenging system with NADPH-dependent reducing activity exists, including aldehyde reductase. Junichi Fujii and Naoyuki Taniguchi, Dysfunction of Redox System by Reactive Oxygen Species, Nitric Oxide and the Glycation Reaction: A Possible Mechanism for Apoptotic Cell Death (Poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996 Dec. 9-20 Riken, Tsukuba, Japan. See, also: Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815.; Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, et al. 1994. Suppression of antioxidative enzyme expression by transforming growth factor-b1 in rat hepatocytes J. Biol. Chem. 269: 15488-15492.; Rosen D R, Siddique T, Patterson D, Figlewicz D A, Sapp P, et al. 1993. Mutations in Cu/Zn-superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362: 59-62.; Fujii J, Myint T, Seo H G, Kayanoki Y, Ikeda Y, et al. 1995. Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells. J. Neurochem. 64: 1456-1461.; Deng H-X, Hentati A, Tainer J A, Iqbal Z, Cayabyab A, et al. 1993. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science 261:1047-1051.; Rothstein J D, Bristol L A, Hosier B, Brown R H, Jr, Kuncl R W. 1994. Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons. Proc. Natl. Acad. Sci. U.S.A. 91: 4155-4159.; Gurnery M E, Pu H, Chiu A Y, Dal Canto, M C, Polchow C Y, et al. 1994. Motor neuron degradation in mice that express a human Cu,Zn-superoxide dismutase mutation. Science 264:1772-1775.; Hockenbery D M, Oltvai Z N, Yin X-M, Milliman C L, Korsmeyer S J. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241-251.; Kayanoki Y, Fujii J, Islam K N, Suzuki K, Kawata S, et al. 1996. The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species. J. Biochem. 119: 817-822.; Islam K N, Kayanoki Y, Kaneto H, Suzuki K, Asahi M, et al. 1996. TGF-b1 triggers oxidative modifications and enhances apoptosis in HIT cells through accumulation of reactive oxygen species by suppression of catalase and glutathione peroxidase. Free Radic. Biol. Med. in press.; Taniguchi N. 1992. Clinical significances of superoxide dismutases: Changes in aging, diabetes, ischemia, and cancer. Adv. Clin. Chem. 29: 1-59.; Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, et al. 1987. Glycation and inactivation of human Cu—Zn-superoxide dismutase. Identification of the in vitro glycation sites. J. Biol. Chem. 262:16969-16972.; Ookawara T, Kawamura N, Kitagawa Y, Taniguchi N. 1992. Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species. J. Biol. Chem. 267:18505-18510.; Fujii J, Mint T, Okado A, Kaneto H, Taniguchi N. 1996. Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components. Nephrol. Dial. Transplant (Supple 19) in press.; Kaneto H, Fujii J, Myint T, Islam K N, Miyazawa N, et al. 1996. Reducing sugar triggers oxidative modification and apoptosis in pancreatic b-cells by provoking oxidative stress through glycation reaction. Biochem. J. in press.; Okado A, Kawasaki Y, Hasuike Y, Takahashi M, Teshima T, et al. 1996. Induction of apoptotic cell death by methylglyoxal and 3-deoxyglucosone in macrophage-derived cell lines. Biochem. Biophys. Res. Commun. 225: 219-224.; Takahashi M, Fujii J, Teshima T, Suzuki K, Shiba T, et al. 1995. Identity of a major 3-deoxyglucosone-reducing enzyme with aldehyde reductase in rat liver established by amino acid sequencing and cDNA expression. Gene 127: 249-253.; Takahashi M, Lu Y, Myint T, Fujii J, Wada Y, et al. 1995. In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme. Identification of glycation sites. Biochemistry 34: 1433-1438.; Takahashi M, Fujii J, Miyoshi E, Hoshi A, Taniguchi N. 1996. Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: Implication in detoxification of cytotoxic aldehydes. Int. J. Cancer. 87: 337-341.; Seo H G, Takata I, Nakamura M, Tatsumi H, Suzuki K, et al. 1995. Induction of nitric oxide synthase and concomitant suppression of superoxide dismutases in experimental colitis in rats. Arch. Biochem. Biophys. 324: 41-47.; Kaneto H, Fujii J, Seo H G, Suzuki K, Matsuoka M, et al. 1995. Apoptotic cell death triggered by nitric oxide in pancreatic b-cells. Diabetes 44: 733-738.; Asahi M, Fujii J, Suzuki K, Seo H G, Kuzuya T, et al. 1995. Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity. J. Biol. Chem. 270: 21035-21039.

Cell-cell adhesion is critical in generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule (VCAM-1; CD 106) are inducible cell surface glycoproteins. The expression of these surface proteins are known to be induced in response to activators such as cytokines (TNF-α, IL-1α & β), PMA, lipopolysaccharide and oxidants. The ligands for ICAM-1 and VCAM-1 on lymphocyte are LFA-1 (CD11a/CD18) and VLA-4, respectively. The inappropriate or abnormal sequestration of leukocytes at specific sites is a central component in the development of a variety of autoimmune diseases and pathologic inflammatory disorders. Focal expression of ICAM-1 have been reported in arterial endothelium overlying early foam cell lesions in both dietary and genetic models of atherosclerosis in rabbits. A role of VCAM-1 in the progression of coronary lesions has also been suggested. Loss or gain of cell surface molecules is thought to determine the mobilization, emigration and invasiveness of epithelial cancer cells. Monocytes from patients with diabetes mellitus are known to have increased adhesion to endothelial cells in culture. Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. Antioxidants can block induced adhesion molecule expression and cell-cell adhesion. Sashwati Roy and Chandan K. Sen, Adhesion Molecules And Cell-Cell Adhesion, packer.berkeley.edu/research/Cell/adhes.

The diabetic will become more susceptible to infections because the immune system approaches collapse when GSH levels fall, analogous to certain defects seen in HIV/AIDS. Peripheral vasculature becomes compromised and blood supply to the extremities is severely diminished because GSH is not available in sufficient amounts to stabilize the nitric oxide (.NO) to effectively exert its vascular dilation (relaxation) property. Gangrene is a common sequel and successive amputations are often the result in later years.

Peripheral neuropathies, the loss of sensation commonly of the feet and lower extremities develop, often followed by aberrant sensations like burning or itching, which can't be controlled. Retinopathy and nephropathy are later events that are actually due to microangiopathy, excessive budding and growth of new blood vessels and capillaries, which often will bleed due to weakness of the new vessel walls. This bleeding causes damage to the retina and kidneys with resulting blindness and renal shutdown, the latter results in required dialysis. Cataracts occur with increasing frequency as the GSH deficiency deepens.

Large and medium sized arteries become sites of accelerated, severe atherosclerosis, with myocardial infarcts at early ages, and of a more severe degree. If diabetics go into heart failure, their mortality rates at one year later are far greater than in non-diabetics. Further, if coronary angioplasty is used to treat their severe atherosclerosis, diabetics are much more likely to have renarrowing of cardiac vessels, termed restenosis.

The above complications are due, in large measure, to GSH deficiency and ongoing free radical reactions. These sequellae frequently and eventually occur despite the use of insulin injections daily that lower blood sugar levels. Good control of blood sugar levels is difficult for the majority of diabetics.

III. Use of GSH to Treat Macular Degeneration

Glutathione may be used to treat Macular Degeneration. The pathology of the disease is described below.

Approximately 1 million people in the United States have significant macular degeneration. One out of every 4 persons aged 55 or above now has macular degeneration and 1 in 2 above the age of 80. As our population ages, this principal cause of blindness in the elderly will increase as well. By the year 2002, 15 million people in the U.S. will suffer from macular degeneration.

Age-related macular degeneration (ARMD) is the disease characterized by either a slow (dry form) or rapid (wet form) onset of destruction and irrevocable loss of rods and cones in the macula of the eye. The macula is the approximate center of the retina wherein the lens of the eye focuses its most intense light. The visual cells, known as the rods and cones, are an outgrowth and active part of the central nervous system. They are responsible and essential for the fine visual discrimination required to see clear details such as faces and facial expression, reading, driving, operation of machinery and electrical equipment and general recognition of surroundings. Ultimately, the destruction of the rods and cones leads to functional, legal blindness. Since there is no overt pain associated with the condition, the first warnings of onset are usually noticeable loss of visual acuity. This may already signal late stage events. It is now thought that one of the very first events in this pathologic process is the formation of a material called “drusen”.

Drusen first appears as either patches or diffuse drops of yellow material deposited upon the surface of the retina in the macula lutea or yellow spot. This is the area of the retina where sunlight is focused by the lens. It is the area of the retina that contains the highest density of rods for acuity. Although cones, which detect color, are lost as well in this disease, it is believed to be loss of rods that causes the blindness. Drusen has been chemically analyzed and found to be composed of a mixture of lipids, much of which are peroxidized by free radical reactions. The Drusen first appears as small collections of material at the base of Bruch's membrane. This produces “bubbles” which push the first layer of cells up off the membrane. Vascular budding, neovascular growth, first appears in these channels.

This first layer of cells is unique. They are retinal pigmented epithelial (RPE) cells and these cells are distantly related to CNS microglia and have a phagocytic function. They are also the layer of cells immediately below the primary retinal cells, the rods and cones. The RPE cells are believed to serve a protective function for the rods and cones since they consume the debris cast off by the rods and cones. It is not known yet whether the pigmented material serves a protective function or is related to phagocytosis only. However, this pigment, although concentrated in organelles, is believed to be composed of peroxidized lipids and melanin.

It is believed, because of the order of events in model systems, that the loss of RPE cells occurs first in ARMD (Age Related Macular Degeneration). Once an area of the retinal macula is devoid of RPE cells, loss of rods, and eventually some cones, occurs. Finally, budding of capillaries begins and we see the typical microangiopathy associated with late stage ARMD. It is also known that RPE cells require large quantities of GSH for their proper functioning. When GSH levels drop severely in these cells, in cell cultures where they can be studied, these cells begin to die. When cultures of these cells are supplemented with GSH in the medium, they thrive. There is increasing evidence that progression of the disease is paced by a more profound deficiency in GSH within the retina and probably within these cells, as indicated by cell culture studies.

It is generally believed that “near” ultraviolet (UVB) and visual light of high intensity primarily from sunlight is a strong contributing factor of ARMD. People with light-colored irises constitute a population at high risk, as do those with jobs that leave them outdoors and in equatorial areas where sunlight is most intense. Additional free radical insults, like smoking, add to the risk of developing ARMD.

Several approaches have been recently tested, including chemotherapy, without success. Currently, there is no effective therapy to treat ARMD. Laser therapy has been developed which has been used widely to slow the damage produced in the slow onset form of the disease by cauterizing neovascular growth. However the eventual outcome of the disease, once it has started to progress, is certain.

Glutathione acts to combat both direct and indirect effects of free radical reactions, as well as altering redox-sensitive gene expression.

IV. Use of GSH for Cellular Regulation with Respect to Reactive Oxygen Species

There are a number of types of messengers carrying signals between cells. One type of messenger which has received significant attention recently are small molecule oxidative or free radical agents, which include reactive oxygen species (ROS). These messengers often act by a non-specific interaction with biological macromolecules which may result in a change in configuration. For example, protein secondary structure is typically controlled by cysteine residues, which are susceptible to oxidation with the formation of disulfide bonds. Oxidization of these bonds forming linkages may result in substantial changes in protein configuration and function.

It has thus become increasingly apparent that O₂ ⁻ and H₂O₂ are signaling molecules, changing the behavior of proteins as diverse as transcription factors and membrane receptors by virtue of their ability to undergo redox reactions with the proteins with which they interact, converting —SH groups to disulfide bonds, for example, and changing the oxidation states of enzyme-associated transition metals. As signaling molecules, O₂ ⁻ and H₂O₂ are manufactured by several types of cells, including fibroblasts, endothelial and vascular smooth muscle cells, neurons, ova, spermatozoa and cells of the carotid body. All these cell types appear to use an NAD(P)H oxidase similar to the classical leukocyte NADPH oxidase to produce these oxidants. The stimuli that elicit oxidant production, however, and the purposes for which the oxidants are employed, vary from cell to cell.

Fibroblasts manufacture small but significant amounts of O₂ ⁻ in response to inflammatory mediators such as N-formylated peptides and interleukin-1. The O₂ ⁻ produced by these cells has been postulated to function as a signaling molecule. Optical spectroscopy has shown that fibroblast membranes contain a heme protein that is different from the flavocytochrome subunit of the leukocyte NADPH oxidase but has properties very similar to those of the leukocyte protein. This heme protein has been suggested as the source of the O₂ ⁻ made by these cells.

Endothelial and vascular smooth muscle cells use an NAD(P)H oxidase to produce O₂ ⁻ in response to angiotensin II, a peptide hormone that increases blood pressure. This increase in blood pressure appears to be due to the consumption by O₂ ⁻ of the NO. that is generated on a continuing basis by the endothelial cells. The resulting fall in NO. concentration raises blood pressure by attenuating or eliminating the vasodilatory effect of NO. that normally prevails in the vascular tree.

Neuronal cells in culture produce oxidants when exposed to amyloid β-peptide, found in amyloid deposits seen in the brains of patients with Alzheimer's disease, or related peptides from other amyloid diseases. The possibility that this O₂ ⁻ is produced by an NADPH oxidase is suggested by the observation that flavoprotein inhibitors known to act on the leukocyte NADPH oxidase also inhibit oxidant production in this system. The production of oxidants may be part of a defense used by the neuron against the peptide, with these oxidants perhaps reacting with the peptide to render it susceptible to proteolytic cleavage.

At the moment of fertilization, a membrane NADPH oxidase in sea urchin ova is activated to produce large amounts of H₂O₂. This oxidant cross-links the proteins of the fertilization membrane by forming dityrosyl bridges, making the membrane impermeable to spermatozoa and thereby preventing polyspermy. This mechanism is common to other species

O₂ ⁻ appears to be necessary for the normal function of spermatozoa. When stimulated by a calcium ionophore, normal spermatozoa generate a 3- to 5-min burst of O₂. The O₂ ⁻ produced in this reaction is involved in capacitation of the spermatozoa, because the acrosomal response to a number of stimuli is suppressed by superoxide dismutase. On the other hand, spermatozoa that produce O₂ ⁻ without stimulation are functionally abnormal, perhaps because of a generalized disruption in their signaling machinery.

The carotid body is a small organ located at the bifurcation of the common carotid artery that measures the oxygen tension of the blood. This organ manufactures H₂O₂ on a continuing basis, and immunological analysis has shown that its cells contain all 4 of the specific subunits of the leukocyte NADPH oxidase, or proteins very closely related to those subunits. It has been postulated that a carotid body NADPH oxidase very similar or identical to the leukocyte NADPH oxidase is a key component of the oxygen-measuring apparatus of the carotid body.

Thus, in addition to phosphorylation as a control mechanism over regulatory protein configuration and function, reactive oxygen species may also play an important role in cellular regulation and signaling. Selective cysteine oxidation-reduction also serves as an important mechanism for post-translational modification of protein function. This mechanism, termed “redox regulation”, has been implicated in a variety of cellular processes such as DNA synthesis, enzyme activation, gene expression, and cell cycle regulation.

Thioredoxin (TRX) is a pleiotropic cellular factor which has thiol-mediated redox activity and plays important roles in regulation of cellular processes, including gene expression. TRX exists either in a reduced, or oxidized form and participates in redox reactions through the reversible oxidation of this active center dithiol. Activity of a number of transcription factors is post-translationally altered by redox modification(s) of specific cysteine residue(s). One such factor is NFκB, whose DNA-binding activity is altered by TRX treatment in vitro. The DNA-binding activity of AP-1 is modified by a DNA repair enzyme, Redox Factor-1 (Ref-1). Ref-1 activity is in turn modified by various redox-active compounds, including TRX. TRX translocates from the cytoplasm into the nucleus in response to PMA treatment to associate directly with Ref-1 and modulates not only the DNA-binding but also the transcriptional activity of the AP-1 molecule.

Human thioredoxin (hTRX) has thus been shown to be an important redox regulator in those biological processes. hTRX can function directly by interacting with the target molecules such as NFκB transcription factor, or indirectly via another redox protein known as redox factor 1 (Ref-1). See, Structural Basis Of Thioredoxin-Mediated Redox-Regulation, Qin et al, (poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996 Dec. 9-20 Riken, Tsukuba, Japan. See, also: Abate, C., Patel, L., Rauscher III, R. J., and Curran, T. (1990) Redox regulation of Fos and Jun DNA binding activity in vitro. Science 249, 1157-1161.; Baeuerle, P. A., and Henkel, T. Function and activation of NFκB in the immune system. (1994) Annu. Rev. Immunol. 12, 141-179.; Bax and Grzesiek, S. (1993) Methodological advances in protein NMR. Accounts Chem. Res. 26, 131-138.; Beg, A. A., and A. S. Baldwin, Jr. The IkB proteins: multifunctional regulators of Rel/NFκB transcription factors. (1993) Genes and Dev. 7, 2064-2070.; Clore, G. M. and Gronenborn, A. M. (1991) Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. Science 252, 1390-1399.; Gilmore, T. D., and Morin, P. J. The IkB proteins: members of a multifunctional family. (1993) Trends Genet. 9, 427-433.; Ghosh, S., van Duyne, G., Ghosh, S., and Sigler, P. Structure of NFκB p50 homodier bound to a kB site. (1995) Nature 373, 303-310.; Hayashi, T., Ueno, Y., and Okamoto, T. Oxidoreductive regulation of nuclear factor kB. (1993) J. Biol. Chem. 268(15)11380-11388.; Holmgren, A. (1989) Thioredoxin and glutaredoxin. J. Biol. Chem. 264, 13963-13966.; Liou, H.-C., and Baltimore, D. Regulation of the NFκB/rel transcription factor and IkB inhibitor system. (1993) Curr. Opin. Cell. Biol. 5, 477-487.; Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. Thioredoxin regulates the DNA binding activity of NFκB by reduction of a disulfide bond involving cysteine 62. (1992) Nucleic Acids Research, 20(15):3821-3830.; Muller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C. Structure of the NFκB p50 homodimer bound to DNA. (1995) Nature 373, 311-317.; Powis, G., Briehl, M., and Oblong, J. (1995) Redox signaling and the control of cell growth and death. Pharmac. Ther. 68, No. 1, 149-173.; Qin, J., Clore, G. M., Kennedy, W M P, Huth, J., and Gronenborn, A. M. (1995) Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NFκB. Structure, 15:3, 289-297.; Qin, J., Clore, G. M., Kennedy, W M P, and Gronenborn, A. M. The solution structure of human thioredoxin complexed with its target from Ref-1 reveals peptide chain reversal. (1996b) Structure, 4(5), 613-620.; Walker, L., Robson, C. N., Black, E., Gillespie, D., and Hickson, I. (1993) Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding. Mol. Cell. Biol. 13, 5370-5376.; Xanthoudakis, S., Miao, G. G., Wang, F., E. Pan, Y., and Curran, T. (1992) Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 11, 653-656.; Xanthoudakis, S., Miao, G. G., and Curran, T. (1994) The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proc. Natl. Acad. Sci. USA, 91, 23-27.

Cellular redox status modulates various aspects of cellular events including proliferation and apoptosis. TRX is a small (13 kDa), ubiquitous protein with two redox-active half-cystine residues in an active center, -Trp-Cys-Gly-Pro-Cys-, and is also known as adult T-cell leukemia-derived factor (ADF) involved in HTLV-I leukemogenesis. The pathway for the reduction of a protein disulfide by TRX entails nucleophilic attack by one of the active-site sulfhydryls to form a protein-protein disulfide followed by intramolecular displacement of the reduced target proteins with concomitant formation of oxidized TRX. Besides the activity as an autocrine growth factor for HTLV-I-infected T cells and Epstein-Barr virus-transformed lymphocytes, numerous studies have shown the importance of ADF/TRX as a cellular reducing catalyst in human physiology.

In vitro and in vivo experiments showed that TRX augmented the DNA-binding and transcriptional activities of the p50 subunit of NFκB by reducing Cys 62 of p50. Direct physical association of TRX and an oligopeptide from NFκB p50 has been revealed by NMR study in vitro. Redox regulation of Jun and Fos molecules has also been implicated. Various antioxidants strongly activate the DNA-binding and transactivation abilities of AP-1 complex. TRX enhances the DNA-binding activity of Jun and Fos, in a process which requires other molecules, such as redox factor-1 (Ref-1).

NFκB regulates expression of a wide variety of cellular and viral genes. These genes include cytokines such as IL-2, IL-6, IL-8, GM-CSF and TNF, cell adhesion molecules such as ICAM-1 and E-selectin, inducible nitric oxidase synthase (iNOS) and viruses such as human immunodeficiency virus (HIV) and cytomegalovirus. Through the causal relationship with these genes, NFκB is considered to be causally involved in the currently intractable diseases such as acquired immunodeficiency syndrome (AIDS), hematogenic cancer cell metastasis and rheumatoid arthritis (RA). Although the genes induced by NFκB are variable according to the context of cell lineage and are also under the control of the other transcription factors, NFκB plays a major role in regulation of these genes and thus contributes a great deal to the pathogenesis. Therefore, biochemical intervention of NFκB should conceivably interfere the pathogenic process and would be effective for the treatment.

NFκB consists of two subunit molecules, p65 and p50, and usually exists as a molecular complex with an inhibitory molecule, Id, in the cytosol. Upon stimulation of the cells such as by proinflammatory cytokines, IL-1 and TNF, Id is dissociated and NFκB is translocated to the nucleus and activates expression of target genes. Thus activity of NFκB itself is regulated by the upstream regulatory mechanism. Not much is know about the upstream signaling cascade. However, there are at least two independent steps in the NFκB activation cascade: kinase pathways and redox-signaling pathway. These two distinct pathways are involved in the NFκB activation cascade in a coordinate fashion, which may contribute to a fine tune, as well as fail-safe, regulation of NFκB activity.

At least two distinct types of kinase pathways are known to be involved in NFκB activation: NFκB kinase and Id kinase. NFκB kinase is a 43 kD serine kinase, associated with NFκB. This kinase phosphorylates both subunits of NFκB and dissociates it from Id. There is another kinase or kinases that is known to phosphorylate Id. Consistent with these findings, NFκB was shown to be phosphorylated in some cell lines and Id was phosphorylated in others in response to stimulation with TNF or IL-1. In most of the cases, NFκB dissociation by kinase cascade is a primary step of NFκB activation.

After dissociation from IKB, however, NFκB must go through the redox regulation by cellular reducing catalyst, thioredoxin (TRX). TRX is known to participate in redox reactions through reversible oxidation of its active center dithiol to a disulfide. Human TRX has been initially identified as a factor responsible for induction of the A subunit of interleukin-2 receptor which is now known to be under the control of NFκB. It is known that NFκB can not bind to the κB DNA sequence of the target genes until it is reduced.

NFκB appears to have a novel DNA-binding structure called beta-barrel, a group of beta sheets stretching toward the target DNA. There is a loop in the tip of the beta barrel structure that intercalates with the nucleotide bases and is considered to make a direct contact with the DNA. This DNA-binding loop contains the cystein 62 residue of NFκB that is likely the target of redox regulation as a proton donor from TRX. A boot-shaped hollow on the surface of TRX containing the redox-active cysteines could stably recognize the DNA-binding loop of p50 and is likely to reduce the oxidized cysteine by donating protons in a structure-dependent way. Therefore, the reduction of NM by TRX is considered to be specific.

Not much is known about the initiation of the NFκB signaling cascades. However, pretreatment of cells with antioxidants such as N-acetyl-cysteine (NAC) or a-lipoic acid blocks NFκB. NAC can also block the induction of TRX. Therefore, anti-NFκB actions of antioxidants are considered to be two-fold: 1) blocking the signaling immediately downstream of the signal elicitation, and 2) suppression of induction of the redox effector TRX. It is noted that, in mammals without chronic diseases, such as HIV infection, diabetes, etc, which might impair physiologic glutathione metabolism, a strategy for the pharmaceutical administration of other antioxidants which improve glutathione metabolism or compounds which are themselves appropriate antioxidants may be employed. It is noted that NAC has been shown to have certain neurological toxicity in chronic administration, and therefore this compound is likely inappropriate. On the other hand, lipoic acid may be an advantageous antioxidant alone, or in combination with glutathione. Because of the sensitivity of glutathione oral administration to the particular method of administration, α-lipoic acid may have to be administered separately.

The intracellular redox cascade involves successive reduction of oxygen by addition of four electrons and redox regulation of a target protein. Among these ROI hydrogen peroxide has a longest half-life and is considered to be a mediator of oxidative signal. On the other hand, cellular reducing system such as TRX counteracts the action of hydrogen peroxide. The intensity of the oxidative signal may be modulated by the internal GSH level. Similarly, total GSH/GSSG content may influence the responsiveness of the cellular redox signaling. Therefore, intracellular cycteine required to produce GSH. See: Holmgren, A. Ann. Rev. Biochem. 54, 237-271 (1985).; Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J. & Hay, R. T. Nucleic Acids Res. 20, 3821-30 (1992).; Okamoto, T., et al. Int. Immunol. 4, 811-9 (1992).; Abate, C., Patel, L., Rauscher, F. J. & Curran, T. Science 249, 1157-61 (1990).; Xanthoudakis, S. & Curran, T. Methods Enzymol. 234, 163-74 (1994).; Xanthoudakis, S. & Curran, T. EMBO J. 11, 653-65 (1992).; Pahl, H. L. & Baeuerle, P. A. BioEssays 16, 497-502 (1994).; Holmgren, A. J. biol. Chem. 264, 13963-1366 (1989).; Tagaya, Y., et al. EMBO J. 8, 757-764 (1989).; Yodoi, J. & Uchiyama, T. Immunol. Today 13, 405-11 (1992).; Silberstein, D. S., McDonough, S., Minkoff, M. S. & Balcewicz Sablinska, M. K. J. biol. Chem. 268, 9138-42 (1993).; Iwata, S., et al. J. Immunol. 152, 5633-42 (1994).; Biguet, C., et al. J. biol. Chem. 269, 28865-70 (1994).; Qin, J., Clore, G. M., Kennedy, W. M. P., Huth, J. R. & Gronenborn, A. M. Structure 3, 289-297 (1995).; Meyer, M., Schreck, R. & Baeuerle, P. A. EMBO J. 12, 2005-2015 (1993).; Xanthoudakis, S., Miao, G. G. & Curran, T. Proc. natl. Acad. Sci. U.S.A. 91, 23-7 (1994).; Isoda, K. & Nüsslein-Volhard, C. Proc. natl. Acad. Sci. U.S.A. 91, 5350-5354 (1994).; Kishigami, S., Kannaya, E., Kikuchi, M. & Ito, K. J. biol. Chem. 270, 17072-17074 (1995).; Oblong, J. E., Berggren, M., Gasdaska, P. Y. & Powis, G. J. biol. Chem. 269, 11714-20 (1994).; Tonissen, K., et al. J. biol. Chem. 268, 22485-9 (1993).; Forman Kay, J. D., Clore, G. M. & Gronenborn, A. M. Biochemistry 31, 3442-52 (1992).; Sadowski, I. & Ptashne, M. Nucleic Acids Res. 17, 7539 (1989).; Perlmann, T., Rangarajan, P. N., Umesono, K. & Evans, R. M. Genes & Develop. 7, 1411-1422 (1993).; Angel, P., et al. Mol. Cell. Biol. 7, 2256-2266 (1987).; Barzilay, G. & Hichson, I. D. BioEssays 17, 713-719 (1995).; Okuno, H., et al. Oncogene 8, 695-701 (1993).; Chida, K. & Vogt, P. K. Proc. natl. Acad. Sci. U.S.A. 89, 4290-4294 (1992).; Ng, L., Forrest, D. & Curran, T. Nucleic Acids Res. 21, 5831-7 (1993).; Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Gene 77, 51-59 (1989).; Nerlov, C. & Ziff, E. B. EMBO J. 14, 4318-4328 (1995).

Membrane receptors and transporters, including, for example, the insulin receptor and receptors for certain neurotransmitters, are regulated by the redox state of the cell. A very large number of enzymes are also regulated by the cell's redox state. A partial list of proteins whose function is regulated by oxidation-reduction is presented in Table 1. Babior, B. M. (1997). “Superoxide: a two-edged sword”, Braz. J. Med. Biol. Res., 30, 141-155.

TABLE 1 Some proteins whose function is regulated by the redox state of the cell. Enzymes Collagenase (146, 147) p21Ras guanine nucleotide-binding protein (148) Protein tyrosine phosphatase (149) p56Lck protein tyrosine kinase (150) Glycogen phosphorylase phosphatase (151) Glycogen synthase (151) Phosphofructokinase (151) Fructose-1,6-bisphosphatase (151) Hexokinase (151) Pyruvate kinase (151, 152) Glucose-6-phosphate dehydrogenase (151) 3-Hydroxy-3-methylglutaryl CoA reductase (151) Serotonin N-acetyltransferase (151) Guanylate cyclase (151) Medium-chain fatty acyl CoA dehydrogenase (153) Xanthine dehydrogenase (154) Chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase (155) Chloroplast NADP-linked malate dehydrogenase (155) Chloroplast sedoheptulose bisphosphatase (155) Fructose bisphosphatase (155) NADP-malic enzyme (156) 3a-Hydroxysteroid dehydrogenase (157) DsbA protein disulfide isomerase from E. coli (158) Creatine kinase (152) Sarcoplasmic reticulum Ca2+-ATPase (152) Transcription factors NF-kappa B (128-130) AP-1 (jun/fos) (131) SoxR (132, 133) SoxS (134) OxyR (135) Hypoxia-inducible factor 1 (159) Thyroid transcription factor I (160) Glucocorticoid receptor (161) Sp1 (161, 162) Receptors NMDA receptor (163) Insulin receptor NMDA receptor (164, 165) Ryanodine receptor (166) HoxB5 (167) c-Myb (167, 168) v-Rel (167) p53 (169) IsI-1 (170)

Others

Erythropoietin RNA-binding protein (171)

-   1. Elstner E F (1990). Der Sauerstoff. Biochemie, Biologie, Medizin.     BI Wissenschaftsverlag, Mannheim/Wien/Zürich. -   2. Fridovich I (1995). Superoxide radical and superoxide dismutases.     Annual Review of Biochemistry, 64: 97-112. -   3. Halliwell B & Gutteridge J M C (1986). Iron and free radical     reactions: two aspects of antioxidant protection. Trends in     Biochemical Sciences, 11: 372-375. -   4. Bielski B H (1985). Fast kinetic studies of dioxygen-derived     species and their metal complexes. Philosophical Transactions of the     Royal Society of London, Series B. Biological Sciences, 311:     473-482. -   5. Goldstein S & Czapski G (1986). The role and mechanism of metal     ions and their complexes in enhancing damage in biological systems     or in protecting these systems from the toxicity of O2-. Free     Radical Biology and Medicine, 2: 3-11. -   6. Harris L R, Cake M H & Macey D J (1994). Iron release from     ferritin and its sensitivity to superoxide ions differs among     vertebrates. Biochemical Journal, 301: 385-389. -   7. Gardner P R, Rainer I, Epstein L B & White C W (1995). Superoxide     radical and iron modulate aconitase activity in mammalian cells.     Journal of Biological Chemistry, 270:13399-13405. -   8. Khan A U & Kasha M (1994). Singlet molecular oxygen in the     Haber-Weiss reaction. Proceedings of the National Academy of     Sciences, USA, 91:12365-12367. -   9. Radi R, Beckman J S, Bush K M & Freeman B A (1991). Peroxynitrite     oxidation of sulfhydryls. The cytotoxic potential of superoxide and     nitric oxide. Journal of Biological Chemistry, 266: 4244-4250. -   10. Kong S-K, Yim M B, Stadtman E R & Chock P B (1996).     Peroxynitrite disables the tyrosine phosphorylation regulatory     mechanism: Lymphocyte-specific tyrosine kinase fails to     phosphorylate nitrated cdc2(6-20)NH2 peptide. Proceedings of the     National Academy of Sciences, USA, 93: 3377-3382. -   11. Winterbourn C C (1985). Comparative reactivities of various     biological compounds with myeloperoxidase-hydrogen     peroxide-chloride, and similarity of the oxidant to hypochlorite.     Biochimica et Biophysica Acta, 840: 204-210. -   12. Thomas E L, Jefferson M M & Grisham M (1982).     Myeloperoxidase-catalyzed incorporation of amino acids into     proteins: Role of hypochlorous acid and chloramines. Biochemistry,     21: 6299-6308. -   13. Grisham M B, Jefferson M M, Melton D F & Thomas E L (1984).     Chlorination of endogenous amines by isolated neutrophils.     Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities     of the chloramines. Journal of Biological Chemistry, 259:     10404-10413. -   14. Kanofsky J R, Hoogland H, Wever R & Weiss S J (1988). Singlet     oxygen production by human eosinophils. Journal of Biological     Chemistry, 263: 9692-9696. -   15. Steinbeck M J, Khan A U, Karnovsky M J & Hegg G G (1992).     Intracellular singlet oxygen generation by phagocytosing neutrophils     in response to particles coated with a chemical trap. Journal of     Biological Chemistry, 267:13425-13433. -   16. McCord J M & Fridovich I (1969). Superoxide dismutase. An     enzymic function for erythrocuprein. Journal of Biological     Chemistry, 244: 6049-6055. -   17. Halliwell B & Gutteridge J M C (1989). Free Radicals in Biology     and Medicine. 2nd edn. Oxford University Press, Oxford. -   18. Hassan H M & Fridovich I (1996). Enzymatic defenses against the     toxicity of oxygen and of streptonigrin in Escherichia coli. Journal     of Bacteriology, 129: 1574-1583. -   19. Farr S B, D. Ari R & Touati D (1986). Oxygen-dependent     mutagenesis in Escherichia coli lacking superoxide dismutase.     Proceedings of the National Academy of Sciences, USA, 83: 8268-8272. -   20. Ballzan R, Bannister W H, Hunter G J & Bannister J V (1995).     Escherichia coli iron superoxide dismutase targeted to the     mitochondria of yeast cells protects the cells against oxidative     stress. Proceedings of the National Academy of Sciences, USA, 92:     4219-4223. -   21. Lapinskas P J, Cunningham K W, Liu X F, Fink G R & Culotta V C     (1995). Mutations in PMR1 suppress oxidative damage in yeast cells     lacking superoxide dismutase. Molecular and Cellular Biology, 15:     1382-1388. -   22. Kelner M J & Bagnell R (1990). Alteration of endogenous     glutathione peroxidase, manganese superoxide dismutase, and     glutathione transferase activity in cells transfected with a     copper-zinc superoxide dismutase expression vector: Explanation for     variations in paraquat resistance. Journal of Biological Chemistry,     265:10872-10875. -   23. Yang G, Chan P H, Chen J, Carlson E, Chen S F, Weinstein P,     Epstein C J & Kamii H (1994). Human copper-zinc superoxide dismutase     transgenic mice are highly resistant to reperfusion injury after     focal cerebral ischemia. Stroke, 25:165-170. -   24. Reveillaud I, Phillips J, Duyf B, Hilliker A, Kongpachith A &     Fleming J E (1994). Phenotypic rescue by a bovine transgene in a     Cu/Zn superoxide dismutase-null mutant of Drosophila melanogaster.     Molecular and Cellular Biology, 14: 1302-1307. -   25. Imlay J A & Linn S (1988). DNA damage and oxygen radical     toxicity. Science, 240: 1302-1309. -   26. Stadtman E R (1992). Protein oxidation and aging. Science,     257:1220-1224. -   27. Thomas C E, Morehouse L A & Aust S D (1985). Ferritin and     superoxide-dependent lipid peroxidation. Journal of Biological     Chemistry, 260: 3275-3280. -   28. Aikens J & Dix T A (1991). Perhydroxyl radical (HOO.) initiated     lipid peroxidation. The role of fatty acid hydroperoxides. Journal     of Biological Chemistry, 266: 15091-15098. -   29. Shigenaga M K, Gimeno C J & Ames B N (1989). Urinary     8-hydroxy-2.-deoxyguanosine as a biological marker in in vivo     oxidative DNA damage. Proceedings of the National Academy of     Sciences, USA, 86: 9697-9701. -   30. Aruoma 01, Halliwell B, Gazewski E & Dizdaroglu M (1989). Damage     to the bases in DNA induced by hydrogen peroxide and ferric ion     chelates. Journal of Biological Chemistry, 264: 20509-20512. -   31. Demple B & Harrison L (1994). Repair of oxidative damage to DNA:     enzymology and biology. Annual Review of Biochemistry, 63: 915-948. -   32. Birnboim H C & Kanabus-Kaminska M (1985). The production of DNA     strand breaks in human leukocytes by superoxide anion may involve a     metabolic process. Proceedings of the National Academy of Sciences,     USA, 82: 6820-6824 -   33. Zingarelli B, O. Connor M, Wong H, Salzman A L & Szabó C (1996).     Peroxynitrite-mediated DNA strand breakage activates poly-adenosine     diphosphate ribosyl synthetase and causes cellular energy depletion     in macrophages stimulated with bacterial lipopolysaccharide. Journal     of Immunology, 156: 350-358. -   34. Burger R M, Projan S J, Horwitz S B & Peisach J (1986). The DNA     cleavage of iron-bleomycin. Kinetic resolution of strand scission     from base propenal release. Journal of Biological Chemistry, 261:     15955-15959. -   35. Szabó C, Zingarelli B, O. Connor M & Salzman A L (1996). DNA     strand breakage, activation of poly(ADP-ribose) synthetase, and     cellular energy depletion are involved in the cytotoxicity in     macrophages and smooth muscle cells exposed to peroxynitrite.     Proceedings of the National Academy of Sciences, USA, 93:1753-1758. -   36. Stadtman E R & Oliver C N (1991). Metalcatalyzed oxidation of     proteins. Physiological consequences. Journal of Biological     Chemistry, 266: 2005-2008. -   37. Davies K J A, Delsignore M E & Lin S W (1987). Protein damage     and degradation by oxygen radicals. II. Modification of amino acids.     Journal of Biological Chemistry, 262: 9902-9907. -   38. Stadtman E R & Berlett B S (1991). Fenton Chemistry. Amino acid     oxidation. Journal of Biological Chemistry, 266: 17201-17211. -   39. Oliver C N, Starke-Reed P E, Stadtman E R, Liu G J, Carney J M &     Floyd R A (1990). Oxidative damage to brain proteins, loss of     glutamine synthetase activity, and production of free radicals     during ischemia/reperfusion-induced injury to gerbil brain.     Proceedings of the National Academy of Sciences, USA, 87: 5144-5147. -   40. Berlett B S, Friguet B, Yim M B, Chock P B & Stadtman E R     (1996). Peroxynitrite-mediated nitration of tyrosine residues in     Escherichia coli glutamine synthetase mimics adenylation: Relevance     to signal transduction. Proceedings of the National Academy of     Sciences, USA, 93: 1776-1780. -   41. Haddad I Y, Pataki G, Calliani C, Beckman J S & Matalon S     (1994). Quantitation of nitrotyrosine levels in lung sections of     patients and animals with acute lung injury. Journal of Clinical     Investigation, 94: 2407-2413. -   42. Albrich J M, McCarthy C A & Hurst J K (1981). Biological     reactivity of hypochlorous acid: Implications for microbicidal     mechanisms of leukocyte myeloperoxidase. Proceedings of the National     Academy of Sciences, USA, 78: 210-214. -   43. Domigan N M, Charlton T S, Duncan M W, Winterburn C C & Kettle     Ai (1995). Chlorination of tyrosyl residues in peptides by     myeloperoxidase and human neutrophils. Journal of Biological     Chemistry, 270:16542-16548. -   44. Bernofsky C, Bandara B M R, Hinojosa O & Strauss S L (1990).     Hypochlorite-modified adenine nucleotides: Structure, spin-trapping     and formation by activated guinea pig polymorphonuclear leukocytes.     Free Radical Research Communications, 9: 303-315. -   45. Porter N A, Caldwell S E & Mills K A (1995). Mechanisms of free     radical oxidation of unsaturated lipids. Lipids, 30: 277-290. -   46. Halliwell B (1993). The chemistry of free radicals. Toxicology     and Industrial Health, 9: 1-21. -   47. Halliwell B & Chirico S (1993). Lipid peroxidation: Its     mechanism, measurement, and significance. American Journal of     Clinical Nutrition, 57: 715S-725S. -   48. Liu S X, Zhou M, Chen Y, Wen W Y & Sun M J (1996).     Lipoperoxidative injury to macrophages by oxidatively modified low     density lipoprotein may play an important role in foam cell     formation. Atherosclerosis, 121: 55-61. -   49. Haberland M E, Fong D & Cheng L (1988). Malondialdehyde-altered     protein occurs in atheroma of Watanabe heritable hyperlipidemic     rabbits. Science, 241: 215-218. -   50. Weitzman S A & Gordon L I (1990). Inflammation and cancer: Role     of phagocytegenerated oxidants in carcinogenesis. Blood, 76:     655-663. -   51. Floyd R A (1990). Role of oxygen free radicals in carcinogenesis     and brain ischemia. FASEB Journal, 4: 2587-2597. -   52. Miesel R, Kurpisz M & Kroger H (1996). Suppression of     inflammatory arthritis by simultaneous inhibition of nitric oxide     synthase and NADPH oxidase. Free Radical Biology and Medicine, 20:     75-81. -   53. Adelman R, Saul R L & Ames B N (1988). Oxidative damage to DNA:     Relation to species metabolic rate and life span. Proceedings of the     National Academy of Sciences, USA, 85: 2706-2708. -   54. Ames B N, Shigenaga M K & Hagen T M (1993). Oxidants,     antioxidants, and the degenerative diseases of aging. Proceedings of     the National Academy of Sciences, USA, 90: 7915-7922. -   55. Fridovich I (1975). Superoxide dismutases. Annual Review of     Biochemistry, 44:147-159. -   56. Fridovich I (1974). Superoxide dismutases. Advances in     Enzymology and Related Areas of Molecular Biology, 41: 35-97. -   57. Hosier B A & Brown Jr R H (1995). Copper/zinc superoxide     dismutase mutations and free radical damage in amyotrophic lateral     sclerosis. Advances in Neurology, 68:41-46. -   58. Wiedau-Pazos M, Goto J J, Rabizadeh S, Gralla E B, Roe J A, Lee     M K, Valentine J S & Bredesen D E (1996). Altered reactivity of     superoxide dismutase in familial amyotrophic lateral sclerosis.     Science, 271: 515-518. -   59. Deisseroth A & Dounce A L (1970). Catalase: Physical and     chemical properties, mechanism of catalysis, and physiological role.     Physiological Reviews, 50: 319-375. -   60. Michiels C, Raes M, Toussaint O & Remade J (1994). Importance of     Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival     against oxidative stress. Free Radical Biology and Medicine, 17:     235-248. -   61. Gaetani G F, Ferraris A M, Rolfo M, Mangerini R, Arena S &     Kirkman H N (1996). Predominant role of catalase in the disposal of     hydrogen peroxide within human erythrocytes. Blood, 87: 1595-1599. -   62. Cohen H J & Avissar N (1993). Molecular and biochemical aspects     of selenium metabolism and deficiency. Progress in Clinical and     Biological Research, 380: 191-202. -   63. Stadtman T C (1990). Selenium biochemistry. Annual Review of     Biochemistry, 59:111-127. -   64. Burk R F (1990). Protection against free radical injury by     selenoenzymes. Pharmacology and Therapeutics, 45: 383-385. -   65. Flohe L (1988). Glutathione peroxidase. Basic Life Sciences, 49:     663-668. -   66. Chambers I & Harrison P R (1987). A new puzzle in selenoprotein     biosynthesis: selenocysteine seems to be encoded by the .stop.     codon, UGA. Trends in Biochemical Sciences, 12: 255-256. -   67. Cohen H J, Chovaniec M E, Mistretta D & Baker S S (1985).     Selenium repletion and glutathione peroxidase—differential effects     on plasma and red blood cell enzyme activity. American Journal of     Clinical Nutrition, 41: 735-747. -   68. Anonymous (1980). Treatment of glutathione peroxidase deficiency     with vitamin E. Nutrition Reviews, 38: 120-122. -   69. Bigley R, Stankova L, Roos D & Loos J (1980).     Glutathione-dependent dehydroascorbate reduction: a determinant of     dehydroascorbate uptake by human polymorphonuclear leukocytes.     Enzyme, 25: 200-204. -   70. Johnson R A, Baker S S, Fallon J T, Maynard III E P, Ruskin J N,     Wen Z, Ge K & Cohen H J (1981). An occidental case of cardiomyopathy     and selenium deficiency. New England Journal of Medicine, 304:     1210-1212. -   71. Anonymous (1980). Prevention of Keshan cardiomyopathy by sodium     selenite. Nutrition Reviews, 38: 278-279. -   72. Maiorino M, Chu F F, Ursini F, Davies K J A, Doroshow J H &     Esworthy R S (1991). Phospholipid hydroperoxide glutathione     peroxidase is the 18-kDa selenoprotein expressed in human tumor cell     lines. Journal of Biological Chemistry, 266: 7728-7732. -   73. Frei B, England L & Ames B N (1989). Ascorbate is an outstanding     antioxidant in human blood plasma. Proceedings of the National     Academy of Sciences, USA, 86: 6337-6381. -   74. Meister A (1994). Glutathione-ascorbic acid antioxidant system     in animals. Journal of Biological Chemistry, 269: 9397-9400. -   75. Anonymous (1989). Expanding knowledge of ascorbic acid     metabolism. Nutrition Reviews, 47: 360-361. -   76. Levine M (1986). New concepts in the biology and biochemistry of     ascorbic acid. New England Journal of Medicine, 314: 892-902. -   77. Chow C K (1991). Vitamin E and oxidative stress. Free Radical     Biology and Medicine, 11: 215-232. -   78. Sies H & Murphy M E (1991). Role of tocopherols in the     protection of biological systems against oxidative damage. Journal     of Photochemistry and Photobiology. B, Biology, 8: 211-218. -   79. Packer L (1991). Protective role of vitamin E in biological     systems. American Journal of Clinical Nutrition, 53: 1050S-1055S. -   80. Burton G W & Ingold K U (1989). Vitamin E as an in vitro and in     vivo antioxidant. Annals of the New York Academy of Sciences, 570:     7-22. -   81. Koyama K, Takatsuki K & Inoue M (1994). Determination of     superoxide and ascorbyl radicals in the circulation of animals under     oxidative stress. Archives of Biochemistry and Biophysics, 309:     323-328. -   82. Roginsky V A & Stegmann H B (1994). Ascorbyl radical as natural     indicator of oxidative stress: Quantitative regularities. Free     Radical Biology and Medicine, 17:93-103. -   83. Stankova L, Bigley R, Wyss S R & Aebi H (1979). Catalase and     dehydroascorbate reductase in human polymorphonuclear leukocytes     (PMN): possible functional relationship. Experientia, 35: 852-853. -   84. Mukai K, Kohno Y & Ishizu K (1988). Kinetic study of the     reaction between vitamin E radical and alkyl hydroperoxides in     solution. Biochemical and Biophysical Research Communications, 155:     1046-1050. -   85. Liebler D C, Kling D S & Reed D J (1986). Antioxidant protection     of phospholipid bilayers by alpha-tocopherol. Control of     alpha-tocopherol status and lipid peroxidation by ascorbic acid and     glutathione. Journal of Biological Chemistry, 261: 12114-12119. -   86. May J M, Qu Z & Morrow J D (1996). Interaction of ascorbate and     a-tocopherol in resealed human erythrocyte ghosts. Transmembrane     electron transfer and protection from lipid peroxidation. Journal of     Biological Chemistry, 271: 10577-10582. -   87. Mukai K, Nishimura M, Ishizu K & Kitamura Y (1989). Kinetic     study of the reaction of vitamin C with vitamin E radicals     (tocopheroxyls) in solution. Biochimica et Biophysica Acta, 991:     276-279. -   88. Weiss S J, Klein R & Slivka A (1982). Chlorination of taurine by     human neutrophils. Journal of Clinical Investigation, 70: 598-607. -   89. Aruoma O I, Halliwell B, Hoey B M & Butler J (1988). The     antioxidant action of taurine, hypotaurine and their metabolic     precursors. Biochemical Journal, 256: 251-255. -   90. Wright C E, Lin T T, Lin Y Y, Sturman J A & Gaull G E (1985).     Taurine scavenges oxidized chlorine in biological systems. Progress     in Clinical and Biological Research, 179: 137-147. -   91. Weiss S J, Klein R, Slivka A & Wei M (1982). Chlorination of     taurine by human neutrophils. Evidence for hypochlorous acid     generation. Journal of Clinical Investigation, 70: 598-607. -   92. Weiss S J, Lampert M B & Test S T (1983). Long-lived oxidants     generated by human neutrophils: Characterization and bioactivity.     Science, 222: 625-628. -   93. Babior B M, Kipnes R S & Curnutte J T (1973). Biological defense     mechanisms: the production by leukocytes of superoxide, a potential     bactericidal agent. Journal of Clinical Investigation, 52: 741-744. -   94. Curnutte J T & Babior B M (1974). Biological defense mechanisms:     the effect of bacteria and serum on superoxide production by     granulocytes. Journal of Clinical Investigation, 53:1662-1672. -   95. Johnston R B & Newman S L (1977). Chronic granulomatous disease.     Pediatric Clinics of North America, 24: 365-376. -   96. Anonymous (1991). A controlled trial of interferon gamma to     prevent infection in chronic granulomatous disease. The     International Chronic Granulomatous Disease Cooperative Study Group.     New England Journal of Medicine, 324: 509-516. -   97. Chanock Si, El Benna J, Smith R M & Babior B M (1994). The     respiratory burst oxidase. Journal of Biological Chemistry, 269:     24519-24522. -   98. Thomas E L & Fishman M (1986). Oxidation of chloride and     thiocyanate by isolated leukocytes. Journal of Biological Chemistry,     261:9694-9702. -   99. She Z-W, Wewers M D, Herzyk D J, Sagone A L & Davis W B (1989).     Tumor necrosis factor primes neutrophils for hypochlorous acid     production. American Journal of Physiology, 257: L338-L345. -   100. Raschke P, Becker B F, Leipert B, Schwartz L M, Zahler S &     Gerlach E (1993). Postischemic dysfunction of the heart induced by     small numbers of neutrophils via formation of hypochlorous acid.     Basic Research in Cardiology, 88: 321-339. -   101. Harrison J E & Schultz J (1976). Studies on the chlorinating     activity of myeloperoxidase. Journal of Biological Chemistry, 251:     1371-1374. -   102. Thomas E L, Bozeman P M, Jefferson M M & King C C (1995).     Oxidation of bromide by the human leukocyte enzymes myeloperoxidase     and eosinophil peroxidase. Formation of bromamines. Journal of     Biological Chemistry, 270: 2906-2913 -   103. Weiss S J, Test S T, Eckman n C M, Roos D & Regiani S (1986).     Brominating oxidants generated by human eosinophils. Science, 234:     200-202. -   104. Thomas E L, Grisham M B & Jefferson M M (1983).     Myeloperoxidase-dependent effect of amines on functions of isolated     neutrophils. Journal of Clinical Investigation, 72: 441-454. -   105. Rosen H, Orman J, Rakita R M, Michel B R & Van Devanter D R     (1990). Loss of DNA-membrane interactions and cessation of DNA     synthesis in myeloperoxidase-treated Escherichia coli. Proceedings     of the National Academy of Sciences, USA, 87:10048-10052. -   106. Larrocha C, deCastro M F, Fontan G, Viloria A, Ferrandoz Chacon     J L & Jimenez C (1982). Hereditary myeloperoxidase deficiency: a     study of 12 cases. Scandinavian Journal of Haematology, 29: 389-397. -   107. Parry M F, Root R K, Metcalf J A, Delaney K K, Kaplow L S &     Richar W J (1981). Myeloperoxidase deficiency: Prevalence and     clinical significance. Annals of Internal Medicine, 95: 293-301. -   108. Omar B A, Gad N M, Jordan M C, Striplin S P, Russell W J,     Downey J M & McCord J M (1990). Cardioprotection by Cu,Zn superoxide     dismutase is lost at high doses in the reoxygenated heart. Free     Radical Biology and Medicine, 9: 465-471. -   109. Omar B A & McCord J M (1990). The cardioprotective effect of     Mn-superoxide dismutase is lost at high doses in the postischemic     isolated rabbit heart. Free Radical Biology and Medicine, 9:     473-478. -   110. Scott M D, Mesh nick S R & Eaton J W (1989). Superoxide     dismutase amplifies organismal sensitivity to ionizing radiation.     Journal of Biological Chemistry, 264: 2498-2501. -   111. Scott M D, Mesh nick S R & Eaton J W (1987). Superoxide     dismutase-rich bacteria. Paradoxical increase in oxidant toxicity.     Journal of Biological Chemistry, 262: 3640-3645. -   112. Winterbourn C C (1981). Cytochrome c reduction by semiquinone     radicals can be indirectly inhibited by superoxide dismutase.     Archives of Biochemistry and Biophysics, 209:159-167. -   113. Cadenas E (1989). Biochemistry of oxygen toxicity. Annual     Review of Biochemistry, 58: 79-110. -   114. Forage R G & Foster M A (1979). Resolution of the coenzyme     B-12-dependent dehydratases of Klebsiella sp. and Citrobacter     freundii. Biochimica et Biophysica Acta, 569: 249-258. -   115. Meier B, Cross A R, Hancock J T, Kaup F J & Jones O T G (1991).     Identification of a superoxide-generating NADPH oxidase system in     human fibroblasts. Biochemical Journal, 275: 241-245. -   116. Meier B, Radeke H H, Selle S, Habermehl G G, Resch K & Sies H     (1990). Human fibroblasts release low amounts of reactive oxygen     species in response to the potent phagocyte stimulants, serumtreated     zymosan, N-formyl-methionylleucyl-phenylalanine, leukotriene B4 or     12-O-tetradecanoylphorbol 13-acetate. Biological Chemistry     Hoppe-Seyler, 371:1021-1025. -   117. Meier B, Radeke H H, Selle S, Younes M, Sies H, Resch K &     Habermehl G G (1989). Human fibroblasts release reactive oxygen     species in response to interleukin-1 or tumour necrosis factor-a.     Biochemical Journal, 263: 539-545. -   118. Schreck R, Meier B, Mannel D N, Droge W & Baeuerle P A (1992).     Dithiocarbamates as potent inhibitors of nuclear factor kappa B     activation in intact cells. Journal of Experimental Medicine, 175:     1181-1194. -   119. Griendling K K, Minieri C A, Ollerenshaw J D & Alexander R W     (1994). Angiotensin II stimulates NADH and NADPH oxidase activity in     cultured vascular smooth muscle cells. Circulation Research,     74:1141-1148. -   120. Rajagopalan S, Surz S, Munzel T, Tarpey M, Freeman B A,     Griendling K K & Harrison D G (1996). Angiotensin II-mediated     hypertension in the rat increases vascular superoxide production via     membrane NADH/NADPH oxidase activation. Contribution to alterations     of vasomotor tone. Journal of Clinical Investigation, 97:1916-1923. -   121. Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Y &     Kimura H (1995). Amyloid peptides are toxic via a common oxidative     mechanism. Proceedings of the National Academy of Sciences, USA, 92:     1989-1993. -   122. Heinecke J W & Shapiro B M (1989). Respiratory burst oxidase of     fertilization. Proceedings of the National Academy of Sciences, USA,     86: 1259-1263. -   123. Aitken R J & Clarkson J S (1987). Cellular basis of defective     sperm function and its association with the genesis of reactive     oxygen species by human spermatozoa. Journal of Reproduction and     Fertility, 81: 459-469. -   124. De Lamirande E, Eiley D & Gagnon C (1993). Inverse relationship     between the induction of human sperm capacitation and spontaneous     acrosome reaction by various biological fluids and the superoxide     scavenging capacity of these fluids. International Journal of     Andrology, 16: 258-266. -   125. Acker H, Bolling B, Delpiano M A, Dufau E, Gorlach A &     Holtermann G (1992). The meaning of H₂O₂ generation in carotid body     cells for pO2 chemoreception. Journal of the Autonomic Nervous     System, 41:41-51. -   126. Cross A R, Henderson L, Jones O T G, Delpiano M A, Hentschel J     & Acker H (1990). Involvement of an NAD(P)H oxidase as a pO2 sensor     protein in the rat carotid body. Biochemical Journal, 272: 743-747. -   127. Kummer W & Acker H (1995). Immunohistochemical demonstration of     four subunits of neutrophil NAD(P)H oxidase in type I cells of     carotid body. Journal of Applied Physiology, 78:1904-1909. -   128. Schreck R, Rieber P & Baeuerle P A (1991). Reactive oxygen     intermediates as apparently widely used messengers in the activation     of the NF-kB transcription factor and HIV-1. EMBO Journal, 10:     2247-2258. -   129. Menon S D, Quin S, Guy G R & Tan Y H (1993). Differential     induction of nuclear NF-kB by protein phosphatase inhibitors in     primary and transformed human cells. Requirement for both oxidation     and phosphorylation in nuclear translocation. Journal of Biological     Chemistry, 268: 26805-26812. -   130. Baeuerle P A & Henkel T (1994). Function and activation of     NF-kappaB in the immune system. Annual Review of Immunology,     12:141-179. -   131. Puri P L, Avantaggiati M L, Burgio V L, Chirillo P, Collepardo     D, Natoli G, Balsano C & Levrero M (1995). Reactive oxygen     intermediates mediate angiotensin II-induced c-Jun.c-Fos heterodimer     DNA binding activity and proliferative hypertrophic responses in     myogenic cells. Journal of Biological Chemistry, 270: 22129-22134. -   132. Park S J & Gunsalus R P (1995). Oxygen, iron, carbon, and     superoxide control of the fumarase fumA and fumC genes of     Escherichia coli: Role of the arcA, fnr, and soxR gene products.     Journal of Bacteriology, 177: 6255-6262. -   133. Hidalgo E & Demple B (1996). Activation of SoxR-dependent     transcription in vitro by noncatalytic or NifS-mediated assembly of     [2Fe-2S] clusters in Apo-SoxR. Journal of Biological Chemistry, 271:     7269-7272. -   134. Jair K W, Fawcett W P, Fujita N, Ishihama A & Wolf Jr R E     (1996). Ambidextrous transcriptional activation by SoxS: Requirement     for the C-terminal domain of the RNA polymerase alpha subunit in a     subset of Escherichia coli superoxide-inducible genes. Molecular     Microbiology, 19: 307-317. -   135. Christman M F, Storz G & Ames B N (1989). OxyR, a positive     regulator of hydrogen peroxide-inducible genes in Escherichia coli     and Salmonella typhimurium, is homologous to a family of bacterial     regulatory proteins. Proceedings of the National Academy of     Sciences, USA, 86: 3484-3488. -   136. Marin-Hincapie M & Garofalo R S (1995). Drosophila insulin     receptor: lectin-binding properties and a role for     oxidativereduction of receptor thiols in activation. Endocrinology,     136: 2357-2366. -   137. Pan Z H, Bahring R, Grantyn R & Lipton S A (1995). Differential     modulation by sulfhydryl redox agents and glutathione of GABA- and     glycine-evoked currents in rat retinal ganglion cells. Journal of     Neuroscience, 15: 1384-1391. -   138. Staal F J T, Anderson M T, Staal G E J, Herzenberg L A & Gitler     C (1994). Redox regulation of signal transduction: Tyrosine     phosphorylation and calcium influx. Proceedings of the National     Academy of Sciences, USA, 91: 3619-3622. -   139. Hidalgo E, Bollinger Jr J M, Bradley T M, Walsh C T & Demple B     (1995). Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR     protein and role of the metal centers in transcription. Journal of     Biological Chemistry, 270: 20908-20914. -   140. Flint D H, Tuminello J F & Emptage M H (1993). The inactivation     of Fe—S cluster containing hydrolases by superoxide. Journal of     Biological Chemistry, 268: 22369-22376. -   141. Halliwell B (1992). Switches in enzymes. Nature, 354:     191-192.142. Bandyopadhyay S & Gronostajski R M (1994).     Identification of a conserved oxidation-sensitive cysteine residue     in the NF1 family of DNA-binding proteins. Journal of Biological     Chemistry, 269: 29949-29955. -   143. Landgraf W, Regulla S, Meyer H E & Hofmann F (1991). Oxidation     of cysteines activates cGMP-dependent protein kinase. Journal of     Biological Chemistry, 266: 16305-16311. -   144. Hayashi T, Ueno Y & Okamoto T (1993). Oxidoreductive regulation     of nuclear factor kappa B: Involvement of a cellular reducing     catalyst thioredoxin. Journal of Biological Chemistry, 268:     11380-11388. -   145. Petronilli V, Constantini P, Scorrano L, Colonna R, Passamonti     S & Bernardi P (1994). The voltage sensor of the mitochondrial     permeability transition pore is tuned by the oxidation-reduction     state of vicinal thiols. Journal of Biological Chemistry, 269:     16638-16642. -   146. Weiss S J, Peppin G, Ortiz X, Ragsdale C & Test S T (1985).     Oxidative autoactivation of latent collagenase by human neutrophils.     Science, 227: 747-749. -   147. Weiss S J & Peppin G J (1986). Collagenolytic metalloenzymes of     the human neutrophil. Characteristics, regulation and potential     function in vivo. Biochemical Pharmacology, 35: 3189-3197. -   148. Lander H M, Ogiste J S, Teng K K & Novogrodsky A (1995). p21ras     as a common signaling target of reactive free radicals and cellular     redox stress. Journal of Biological Chemistry, 270: 21195-21198. -   149. Fialkow L, Chan C K, Grinstein S & Downey G P (1993).     Regulation of tyrosine phosphorylation in neutrophils by the NADPH     oxidase. Role of reactive oxygen intermediates. Journal of     Biological Chemistry, 268:17131-17137. -   150. Hardwick J S & Sefton M B (1995). Activation of the Lck     tyrosine protein kinase by hydrogen peroxide requires the     phosphorylation of Tyr-394. Proceedings of the National Academy of     Sciences, USA, 92: 4527-4531. -   151. Ziegler D M (1985). Role of reversible oxidation-reduction of     enzyme thiols-disulfides in metabolic regulation. Annual Review of     Biochemistry, 54: 305-329. -   152. Korge P & Campbell K B (1993). The effect of changes in iron     redox state on the activity of enzymes sensitive to modification of     SH groups. Archives of Biochemistry and Biophysics, 304: 420-428. -   153. Johnson B D, Mancini-Samuelson G J & Stankovich M T (1995).     Effect of transition-state analogues on the redox properties of     medium-chain acyl-CoA dehydrogenase. Biochemistry, 34: 7047-7055. -   154. Hassoun P M, Yu F S, Zulueta J J, White A C & Lanzillo J J     (1995). Effect of nitric oxide and cell redox status on the     regulation of endothelial cell xanthine dehydrogenase. American     Journal of Physiology, 268: L809-L817. -   155. Li D, Stevens F J, Schiffer M & Anderson L E (1994). Mechanism     of light modulation: Identification of potential redox-sensitive     cysteines distal to catalytic site in light-activated chloroplast     enzymes. Biophysical Journal, 67: 29-35. -   156. Drincovich M F & Andreo C S (1994). Redox regulation of maize     NADP-malic enzyme by thiol-disulfide interchange: effect of reduced     thioredoxin on activity. Biochimica et Biophysica Acta, 1206: 10-16. -   157. Terada T, Nanjo H, Shinagawa K, Umemura T, Nishinaka T,     Mizoguchi T & Nishihara T (1993). Modulation of 3 a-hydroxysteroid     dehydrogenase activity by the redox state of glutathione. Journal of     Enzyme Inhibition, 7: 33-41. -   158. Wunderlich M, Jaenicke R & Glockshuber R (1993). The redox     properties of protein disulfide isomerase (DsbA) of Escherichia coli     result from a tense conformation of its oxidized form. Journal of     Molecular Biology, 233: 559-566. -   159. Wang G L, Jiang B H & Semenza G L (1995). Effect of altered     redox states on expression and DNA-binding activity of     hypoxia-inducible factor 1. Biochemical and Biophysical Research     Communications, 212: 550-556. -   160. Arnone M I, Zannini M & Di Lauro R (1995). The DNA binding     activity and the dimerization ability of the thyroid transcription     factor I are redox regulated. Journal of Biological Chemistry,     270:12048-12055. -   161. Esposito F, Cuccovillo F, Morra F, Russo T & Cimino F (1995).     DNA binding activity of the glucocorticoid receptor is sensitive to     redox changes in intact cells. Biochimica et Biophysica Acta, 1260:     308-314. -   162. Ammendola R, Mesuraca M, Russo T & Cimino F (1994). The     DNA-binding efficiency of Sp1 is affected by redox changes. European     Journal of Biochemistry, 225: 483-489. -   163. Gozlan H, Khazipov R & Ben-Ari Y (1995). Multiple forms of     long-term potentiation and multiple regulatory sites of     N-methyl-D-aspartate receptors: role of the redox site. Journal of     Neurobiology, 26: 360-369. -   164. Sullivan J M, Traynelis S F, Chen H S, Escobar W, Heinemann S F     & Lipton S A (1994). Identification of two cysteine residues that     are required for redox modulation of NMDA subtype of glutamate     receptor. Neuron, 13: 929-936. -   165. Tang L H & Aizenman E (1993). Longlasting modification of the     N-methyl-Daspartate receptor channel by a voltage dependent     sulfhydryl redox process. Molecular Pharmacology, 44: 473-478. -   166. Liu G & Pessah I N (1994). Molecular interaction between     ryanodine receptor and glycoprotein triadin involves redox cycling     of functionally important hyperreactive sulfhydryls. Journal of     Biological Chemistry, 269: 33028-33034. -   167. Galang C K & Hauser C A (1993). Cooperative DNA binding of the     human HoxB5 (Hox-2.1) protein is under redox regulation in vitro.     Molecular and Cellular Biology, 13: 4609-4617. -   168. Myrset A H, Bostad A, Jamin N, Lirsac P N, Toma F & Gabrielsen     O S (1993). DNA and redox state induced conformational changes in     the DNA-binding domain of the Myb oncoprotein. EMBO Journal, 12:     4625-4633. -   169. Hainaut P & Milner J (1993). Redox modulation of p53     conformation and sequence-specific DNA binding in vitro. Cancer     Research, 53: 4469-4473. -   170. Sanchez-Garcia I & Rabbitts T H (1993). Redox regulation of in     vitro DNA-binding activity by the homeodomain of the Isl-1 protein.     Journal of Molecular Biology, 231: 945-949. -   171. Rondon I J, Scandurro A B, Wilson R B & Beckman B S (1995).     Changes in redox affect the activity of erythropoietin RNA binding     protein. FEBS Letters, 359: 267-270. -   These oxidants generally act by effecting alterations in iron-sulfur     clusters or by inducing the formation or rupture of disulfide bonds     on whose status the function of the protein depends. See, B. M.     Babior, “Superoxide: a two-edged sword”, Braz J Med Biol Res,     February 1997, Volume 30(2) 141-155. See, also: Elstner E F (1990).     Der Sauerstoff. Biochemie, Biologie, Medizin. BI     Wissenschaftsverlag, Mannheim/Wien/Zürich; Fridovich I (1995).     Superoxide radical and superoxide dismutases. Annual Review of     Biochemistry, 64: 97-112.; Halliwell B & Gutteridge J M C (1986).     Iron and free radical reactions: two aspects of antioxidant     protection. Trends in Biochemical Sciences, 11: 372-375.; Bielski B     H (1985). Fast kinetic studies of dioxygen-derived species and their     metal complexes. Philosophical Transactions of the Royal Society of     London, Series B. Biological Sciences, 311: 473-482.; Goldstein S &     Czapski G (1986). The role and mechanism of metal ions and their     complexes in enhancing damage in biological systems or in protecting     these systems from the toxicity of O₂ ⁻ . Free Radical Biology and     Medicine, 2: 3-11.; Harris L R, Cake M H & Macey D J (1994). Iron     release from ferritin and its sensitivity to superoxide ions differs     among vertebrates. Biochemical Journal, 301: 385-389.; Gardner P R,     Rainer I, Epstein L B & White C W (1995). Superoxide radical and     iron modulate aconitase activity in mammalian cells. Journal of     Biological Chemistry, 270:13399-13405.; Khan A U & Kasha M (1994).     Singlet molecular oxygen in the Haber-Weiss reaction. Proceedings of     the National Academy of Sciences, USA, 91: 12365-12367.; Radi R,     Beckman J S, Bush K M & Freeman B A (1991). Peroxynitrite oxidation     of sulfhydryls. The cytotoxic potential of superoxide and nitric     oxide. Journal of Biological Chemistry, 266: 4244-4250.; Kong S-K,     Yim M B, Stadtman E R & Chock P B (1996). Peroxynitrite disables the     tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific     tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH₂     peptide. Proceedings of the National Academy of Sciences, USA, 93:     3377-3382.; Winterbourn C C (1985). Comparative reactivities of     various biological compounds with myeloperoxidase-hydrogen     peroxide-chloride, and similarity of the oxidant to hypochlorite.     Biochimica et Biophysica Acta, 840: 204-210.; Thomas E L, Jefferson     M M & Grisham M (1982). Myeloperoxidase-catalyzed incorporation of     amino acids into proteins: Role of hypochlorous acid and     chloramines. Biochemistry, 21: 6299-6308.; Grisham M B, Jefferson M     M, Melton D F & Thomas E L (1984). Chlorination of endogenous amines     by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic,     and cytolytic activities of the chloramines. Journal of Biological     Chemistry, 259: 10404-10413.; Kanofsky J R, Hoogland H, Wever R &     Weiss Si (1988). Singlet oxygen production by human eosinophils.     Journal of Biological Chemistry, 263: 9692-9696.; Steinbeck M J,     Khan A U, Karnovsky A M & Hegg G G (1992). Intracellular singlet     oxygen generation by phagocytosing neutrophils in response to     particles coated with a chemical trap. Journal of Biological     Chemistry, 267:13425-13433.; McCord J M & Fridovich I (1969).     Superoxide dismutase. An enzymic function for erythrocuprein.     Journal of Biological Chemistry, 244: 6049-6055.; Halliwell B &     Gutteridge J M C (1989). Free Radicals in Biology and Medicine. 2nd     Ed. Oxford University Press, Oxford.; Hassan H M & Fridovich I     (1996). Enzymatic defenses against the toxicity of oxygen and of     streptonigrin in Escherichia coli. Journal of Bacteriology, 129:     1574-1583.; Farr S B, D'Ari R & Touati D (1986). Oxygen-dependent     mutagenesis in Escherichia coli lacking superoxide dismutase.     Proceedings of the National Academy of Sciences, USA, 83:     8268-8272.; Ballzan R, Bannister W H, Hunter G J & Bannister J V     (1995). Escherichia coli iron superoxide dismutase targeted to the     mitochondria of yeast cells protects the cells against oxidative     stress. Proceedings of the National Academy of Sciences, USA, 92:     4219-4223.; Lapinskas P J, Cunningham K W, Liu X F, Fink G R &     Culotta V C (1995). Mutations in PMR1 suppress oxidative damage in     yeast cells lacking superoxide dismutase. Molecular and Cellular     Biology, 15: 1382-1388.; Kelner A M & Bagnell R (1990). Alteration     of endogenous glutathione peroxidase, manganese superoxide     dismutase, and glutathione transferase activity in cells transfected     with a copper-zinc superoxide dismutase expression vector:     Explanation for variations in paraquat resistance. Journal of     Biological Chemistry, 265:10872-10875.; Yang G, Chan P H, Chen J,     Carlson E, Chen S F, Weinstein P, Epstein C J & Kamii H (1994).     Human copper-zinc superoxide dismutase transgenic mice are highly     resistant to reperfusion injury after focal cerebral ischemia.     Stroke, 25:165-170.; Reveillaud I, Phillips J, Duyf B, Hilliker A,     Kongpachith A & Fleming J E (1994). Phenotypic rescue by a bovine     transgene in a Cu/Zn superoxide dismutase-null mutant of Drosophila     melanogaster. Molecular and Cellular Biology, 14: 1302-1307.; Imlay     J A & Linn S (1988). DNA damage and oxygen radical toxicity.     Science, 240: 1302-1309.; Stadtman E R (1992). Protein oxidation and     aging. Science, 257:1220-1224.; Thomas C E, Morehouse L A & Aust S D     (1985). Ferritin and superoxide-dependent lipid peroxidation.     Journal of Biological Chemistry, 260: 3275-3280.; Aikens J & Dix T A     (1991). Perhydroxyl radical (HOO.) initiated lipid peroxidation. The     role of fatty acid hydroperoxides. Journal of Biological Chemistry,     266: 15091-15098.; Shigenaga M K, Gimeno C J & Ames B N (1989).     Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker in in     vivo oxidative DNA damage. Proceedings of the National Academy of     Sciences, USA, 86: 9697-9701.; Aruoma O I, Halliwell B, Gazewski E &     Dizdaroglu M (1989). Damage to the bases in DNA induced by hydrogen     peroxide and ferric ion chelates. Journal of Biological Chemistry,     264: 20509-20512.; Demple B & Harrison L (1994). Repair of oxidative     damage to DNA: enzymology and biology. Annual Review of     Biochemistry, 63: 915-948.; Birnboim H C & Kanabus-Kaminska M     (1985). The production of DNA strand breaks in human leukocytes by     superoxide anion may involve a metabolic process. Proceedings of the     National Academy of Sciences, USA, 82: 6820-6824.; Zingarelli B,     O'Connor M, Wong H, Salzman A L & Szabó C (1996).     Peroxynitrite-mediated DNA strand breakage activates poly-adenosine     diphosphate ribosyl synthetase and causes cellular energy depletion     in macrophages stimulated with bacterial lipopolysaccharide. Journal     of Immunology, 156: 350-358.; Burger R M, Projan S J, Horwitz S B &     Peisach J (1986). The DNA cleavage of iron-bleomycin. Kinetic     resolution of strand scission from base propenal release. Journal of     Biological Chemistry, 261: 15955-15959.; Szabó C, Zingarelli B,     O'Connor M & Salzman A L (1996). DNA strand breakage, activation of     poly(ADP-ribose) synthetase, and cellular energy depletion are     involved in the cytotoxicity in macrophages and smooth muscle cells     exposed to peroxynitrite. Proceedings of the National Academy of     Sciences, USA, 93:1753-1758.; Stadtman E R & Oliver C N (1991).     Metal-catalyzed oxidation of proteins. Physiological consequences.     Journal of Biological Chemistry, 266: 2005-2008.; Davies K J A,     Delsignore M E & Lin S W (1987). Protein damage and degradation by     oxygen radicals. II. Modification of amino acids. Journal of     Biological Chemistry, 262: 9902-9907.; Stadtman E R & Berlett B S     (1991). Fenton Chemistry. Amino acid oxidation. Journal of     Biological Chemistry, 266:17201-17211.; Oliver C N, Starke-Reed P E,     Stadtman E R, Liu G J, Carney J M & Floyd R A (1990). Oxidative     damage to brain proteins, loss of glutamine synthetase activity, and     production of free radicals during ischemia/reperfusion-induced     injury to gerbil brain. Proceedings of the National Academy of     Sciences, USA, 87: 5144-5147.; Berlett B S, Friguet B, Yim M B,     Chock P B & Stadtman E R (1996). Peroxynitrite-mediated nitration of     tyrosine residues in Escherichia coli glutamine synthetase mimics     adenylation: Relevance to signal transduction. Proceedings of the     National Academy of Sciences, USA, 93: 1776-1780.; Haddad I Y,     Pataki G, Calliani C, Beckman J S & Matalon S (1994). Quantitation     of nitrotyrosine levels in lung sections of patients and animals     with acute lung injury. Journal of Clinical Investigation, 94:     2407-2413.; Albrich J M, McCarthy C A & Hurst J K (1981). Biological     reactivity of hypochlorous acid: Implications for microbicidal     mechanisms of leukocyte myeloperoxidase. Proceedings of the National     Academy of Sciences, USA, 78: 210-214.; Domigan N M, Charlton T S,     Duncan M W, Winterburn C C & Kettle Ai (1995). Chlorination of     tyrosyl residues in peptides by myeloperoxidase and human     neutrophils. Journal of Biological Chemistry, 270: 16542-16548.;     Bernofsky C, Bandara B M R, Hinojosa O & Strauss S L (1990).     Hypochlorite-modified adenine nucleotides: Structure, spin-trapping     and formation by activated guinea pig polymorphonuclear leukocytes.     Free Radical Research Communications, 9: 303-315.; Porter N A,     Caldwell S E & Mills K A (1995). Mechanisms of free radical     oxidation of unsaturated lipids. Lipids, 30: 277-290.; Halliwell B     (1993). The chemistry of free radicals. Toxicology and Industrial     Health, 9: 1-21.; Halliwell B & Chirico S (1993). Lipid     peroxidation: Its mechanism, measurement, and significance. American     Journal of Clinical Nutrition, 57: 715S-725S.; Liu S X, Zhou M, Chen     Y, Wen W Y & Sun A M (1996). Lipoperoxidative injury to macrophages     by oxidatively modified low density lipoprotein may play an     important role in foam cell formation. Atherosclerosis, 121: 55-61.;     Haberland M E, Fong D & Cheng L (1988). Malondialdehyde-altered     protein occurs in atheroma of Watanabe heritable hyperlipidemic     rabbits. Science, 241: 215-218.; Weitzman S A & Gordon L I (1990).     Inflammation and cancer: Role of phagocyte-generated oxidants in     carcinogenesis. Blood, 76: 655-663.; Floyd R A (1990). Role of     oxygen free radicals in carcinogenesis and brain ischemia. FASEB     Journal, 4: 2587-2597.; Miesel R, Kurpisz M & Kroger H (1996).     Suppression of inflammatory arthritis by simultaneous inhibition of     nitric oxide synthase and NADPH oxidase. Free Radical Biology and     Medicine, 20:75-81.; Adelman R, Saul R L & Ames B N (1988).     Oxidative damage to DNA: Relation to species metabolic rate and life     span. Proceedings of the National Academy of Sciences, USA, 85:     2706-2708.; Ames B N, Shigenaga M K & Hagen T M (1993). Oxidants,     antioxidants, and the degenerative diseases of aging. Proceedings of     the National Academy of Sciences, USA, 90: 7915-7922.; Fridovich I     (1975). Superoxide dismutases. Annual Review of Biochemistry, 44:     147-159.; Fridovich I (1974). Superoxide dismutases. Advances in     Enzymology and Related Areas of Molecular Biology, 41: 35-97.;     Hosier B A & Brown Jr R H (1995). Copper/zinc superoxide dismutase     mutations and free radical damage in amyotrophic lateral sclerosis.     Advances in Neurology, 68: 41-46.; Wiedau-Pazos M, Goto J J,     Rabizadeh S, Gralla E B, Roe J A, Lee M K, Valentine J S & Bredesen     D E (1996). Altered reactivity of superoxide dismutase in familial     amyotrophic lateral sclerosis. Science, 271: 515-518.; Deisseroth A     & Dounce A L (1970). Catalase: Physical and chemical properties,     mechanism of catalysis, and physiological role. Physiological     Reviews, 50: 319-375.; Michiels C, Raes M, Toussaint O & Remade J     (1994). Importance of Se-glutathione peroxidase, catalase, and     Cu/Zn-SOD for cell survival against oxidative stress. Free Radical     Biology and Medicine, 17: 235-248.; Gaetani G F, Ferraris A M, Rolfo     M, Mangerini R, Arena S & Kirkman H N (1996). Predominant role of     catalase in the disposal of hydrogen peroxide within human     erythrocytes. Blood, 87: 1595-1599.; Cohen H J & Avissar N (1993).     Molecular and biochemical aspects of selenium metabolism and     deficiency. Progress in Clinical and Biological Research, 380:     191-202.; Stadtman T C (1990). Selenium biochemistry. Annual Review     of Biochemistry, 59: 111-127.; Burk R F (1990). Protection against     free radical injury by selenoenzymes. Pharmacology and Therapeutics,     45: 383-385.; Flohe L (1988). Glutathione peroxidase. Basic Life     Sciences, 49: 663-668.; Chambers I & Harrison P R (1987). A new     puzzle in selenoprotein biosynthesis: selenocysteine seems to be     encoded by the “stop” codon, UGA. Trends in Biochemical Sciences,     12: 255-256.; Cohen H J, Chovaniec M E, Mistretta D & Baker S S     (1985). Selenium repletion and glutathione peroxidase—differential     effects on plasma and red blood cell enzyme activity. American     Journal of Clinical Nutrition, 41: 735-747.; Anonymous (1980).     Treatment of glutathione peroxidase deficiency with vitamin E.     Nutrition Reviews, 38: 120-122.; Bigley R, Stankova L, Roos D & Loos     J (1980). Glutathione-dependent dehydroascorbate reduction: a     determinant of dehydroascorbate uptake by human polymorphonuclear     leukocytes. Enzyme, 25: 200-204.; Johnson R A, Baker S S, Fallon J     T, Maynard III EP, Ruskin J N, Wen Z, Ge K & Cohen H J (1981). An     occidental case of cardiomyopathy and selenium deficiency. New     England Journal of Medicine, 304: 1210-1212.; Anonymous (1980).     Prevention of Keshan cardiomyopathy by sodium selenite. Nutrition     Reviews, 38: 278-279.; Maiorino M, Chu F F, Ursini F, Davies K J A,     Doroshow J H & Esworthy R S (1991). Phospholipid hydroperoxide     glutathione peroxidase is the 18-kDa selenoprotein expressed in     human tumor cell lines. Journal of Biological Chemistry, 266:     7728-7732.; Frei B, England L & Ames B N (1989). Ascorbate is an     outstanding antioxidant in human blood plasma. Proceedings of the     National Academy of Sciences, USA, 86: 6337-6381.; Meister A (1994).     Glutathione-ascorbic acid antioxidant system in animals. Journal of     Biological Chemistry, 269: 9397-9400.; Anonymous (1989). Expanding     knowledge of ascorbic acid metabolism. Nutrition Reviews, 47:     360-361.; Levine M (1986). New concepts in the biology and     biochemistry of ascorbic acid. New England Journal of Medicine, 314:     892-902.; Chow C K (1991). Vitamin E and oxidative stress. Free     Radical Biology and Medicine, 11: 215-232.; Sies H & Murphy M E     (1991). Role of tocopherols in the protection of biological systems     against oxidative damage. Journal of Photochemistry and     Photobiology. B, Biology, 8: 211-218.; Packer L (1991). Protective     role of vitamin E in biological systems. American Journal of     Clinical Nutrition, 53: 1050S-1055S.; Burton G W & Ingold K U     (1989). Vitamin E as an in vitro and in vivo antioxidant. Annals of     the New York Academy of Sciences, 570: 7-22.; Koyama K, Takatsuki K     & Inoue M (1994). Determination of superoxide and ascorbyl radicals     in the circulation of animals under oxidative stress. Archives of     Biochemistry and Biophysics, 309: 323-328.; Roginsky V A & Stegmann     H B (1994). Ascorbyl radical as natural indicator of oxidative     stress: Quantitative regularities. Free Radical Biology and     Medicine, 17: 93-103.; Stankova L, Bigley R, Wyss S R & Aebi H     (1979). Catalase and dehydroascorbate reductase in human     polymorphonuclear leukocytes (PMN): possible functional     relationship. Experientia, 35: 852-853.; Mukai K, Kohno Y & Ishizu K     (1988). Kinetic study of the reaction between vitamin E radical and     alkyl hydroperoxides in solution. Biochemical and Biophysical     Research Communications, 155: 1046-1050.; Liebler D C, Kling D S &     Reed D J (1986). Antioxidant protection of phospholipid bilayers by     α-tocopherol. Control of α-tocopherol status and lipid peroxidation     by ascorbic acid and glutathione. Journal of Biological Chemistry,     261: 12114-12119.; May J M, Qu Z & Morrow J D (1996). Interaction of     ascorbate and ?-tocopherol in resealed human erythrocyte ghosts.     Transmembrane electron transfer and protection from lipid     peroxidation. Journal of Biological Chemistry, 271: 10577-10582.;     Mukai K, Nishimura M, Ishizu K & Kitamura Y (1989). Kinetic study of     the reaction of vitamin C with vitamin E radicals (tocopheroxyls) in     solution. Biochimica et Biophysica Acta, 991: 276-279.; Weiss Si,     Klein R & Slivka A (1982). Chlorination of taurine by human     neutrophils. Journal of Clinical Investigation, 70: 598-607.; Aruoma     01, Halliwell B, Hoey B M & Butler J (1988). The antioxidant action     of taurine, hypotaurine and their metabolic precursors. Biochemical     Journal, 256: 251-255.; Wright C E, Lin T T, Lin Y Y, Sturman J A &     Gaull G E (1985). Taurine scavenges oxidized chlorine in biological     systems. Progress in Clinical and Biological Research, 179:     137-147.; Weiss Si, Klein R, Slivka A & Wei M (1982). Chlorination     of taurine by human neutrophils. Evidence for hypochlorous acid     generation. Journal of Clinical Investigation, 70: 598-607.; Weiss S     J, Lampert M B & Test S T (1983). Long-lived oxidants generated by     human neutrophils: Characterization and bioactivity. Science, 222:     625-628.; Babior B M, Kipnes R S & Curnutte J T (1973). Biological     defense mechanisms: the production by leukocytes of superoxide, a     potential bactericidal agent. Journal of Clinical Investigation, 52:     741-744.; Curnutte J T & Babior B M (1974). Biological defense     mechanisms: the effect of bacteria and serum on superoxide     production by granulocytes. Journal of Clinical Investigation, 53:     1662-1672.; Johnston R B & Newman S L (1977). Chronic granulomatous     disease. Pediatric Clinics of North America, 24: 365-376.; Anonymous     (1991). A controlled trial of interferon gamma to prevent infection     in chronic granulomatous disease. The International Chronic     Granulomatous Disease Cooperative Study Group. New England Journal     of Medicine, 324: 509-516.; Chanock S J, El Benna J, Smith R M &     Babior B M (1994). The respiratory burst oxidase. Journal of     Biological Chemistry, 269: 24519-24522.; Thomas E L & Fishman M     (1986). Oxidation of chloride and thiocyanate by isolated     leukocytes. Journal of Biological Chemistry, 261: 9694-9702.; She     Z-W, Wewers M D, Herzyk D J, Sagone A L & Davis W B (1989). Tumor     necrosis factor primes neutrophils for hypochlorous acid production.     American Journal of Physiology, 257: L338-L345.; Raschke P, Becker B     F, Leipert B, Schwartz L M, Zahler S & Gerlach E (1993).     Postischemic dysfunction of the heart induced by small numbers of     neutrophils via formation of hypochlorous acid. Basic Research in     Cardiology, 88: 321-339.; Harrison J E & Schultz J (1976). Studies     on the chlorinating activity of myeloperoxidase. Journal of     Biological Chemistry, 251: 1371-1374.; Thomas E L, Bozeman P M,     Jefferson M M & King C C (1995). Oxidation of bromide by the human     leukocyte enzymes myeloperoxidase and eosinophil peroxidase.     Formation of bromamines. Journal of Biological Chemistry, 270:     2906-2913.; Weiss S J, Test S T, Eckmann C M, Roos D & Regiani S     (1986). Brominating oxidants generated by human eosinophils.     Science, 234: 200-202.; Thomas E L, Grisham M B & Jefferson M M     (1983). Myeloperoxidase-dependent effect of amines on functions of     isolated neutrophils. Journal of Clinical Investigation, 72:     441-454.; Rosen H, Orman J, Rakita R M, Michel B R & VanDevanter DR     (1990). Loss of DNA-membrane interactions and cessation of DNA     synthesis in myeloperoxidase-treated Escherichia coli. Proceedings     of the National Academy of Sciences, USA, 87: 10048-10052.; Larrocha     C, deCastro M F, Fontan G, Viloria A, Ferrandoz Chacon J L & Jimenez     C (1982). Hereditary myeloperoxidase deficiency: a study of 12     cases. Scandinavian Journal of Haematology, 29: 389-397.; Parry M F,     Root R K, Metcalf J A, Delaney K K, Kaplow L S & Richar W J (1981).     Myeloperoxidase deficiency: Prevalence and clinical significance.     Annals of Internal Medicine, 95: 293-301.; Omar B A, Gad N M, Jordan     M C, Striplin S P, Russell W J, Downey J M & McCord J M (1990).     Cardioprotection by Cu,Zn-superoxide dismutase is lost at high doses     in the reoxygenated heart. Free Radical Biology and Medicine, 9:     465-471.; Omar B A & McCord J M (1990). The cardioprotective effect     of Mn-superoxide dismutase is lost at high doses in the postischemic     isolated rabbit heart. Free Radical Biology and Medicine, 9:     473-478.; Scott M D, Meshnick S R & Eaton J W (1989). Superoxide     dismutase amplifies organismal sensitivity to ionizing radiation.     Journal of Biological Chemistry, 264: 2498-2501.; Scott M D,     Meshnick S R & Eaton J W (1987). Superoxide dismutase-rich bacteria.     Paradoxical increase in oxidant toxicity. Journal of Biological     Chemistry, 262: 3640-3645.; Winterbourn C C (1981). Cytochrome c     reduction by semiquinone radicals can be indirectly inhibited by     superoxide dismutase. Archives of Biochemistry and Biophysics,     209:159-167.; Cadenas E (1989). Biochemistry of oxygen toxicity.     Annual Review of Biochemistry, 58: 79-110.; Forage R G & Foster M A     (1979). Resolution of the coenzyme B-12-dependent dehydratases of     Klebsiella sp. and Citrobacter freundii. Biochimica et Biophysica     Acta, 569: 249-258.; Meier B, Cross A R, Hancock J T, Kaup F J &     Jones O T G (1991). Identification of a superoxide-generating NADPH     oxidase system in human fibroblasts. Biochemical Journal, 275:     241-245.; Meier B, Radeke H H, Selle S, Habermehl G G, Resch K &     Sies H (1990). Human fibroblasts release low amounts of reactive     oxygen species in response to the potent phagocyte stimulants,     serum-treated zymosan, N-formyl-methionyl-leucyl-phenylalanine,     leukotriene B4 or 12-O-tetradecanoylphorbol 13-acetate. Biological     Chemistry Hoppe-Seyler, 371:1021-1025.; Meier B, Radeke H H, Selle     S, Younes M, Sies H, Resch K & Habermehl G G (1989). Human     fibroblasts release reactive oxygen species in response to     interleukin-1 or tumour necrosis factor-α. Biochemical Journal, 263:     539-545.; Schreck R, Meier B, Mannel D N, Droge W & Baeuerle P A     (1992). Dithiocarbamates as potent inhibitors of nuclear factor κB     activation in intact cells. Journal of Experimental Medicine, 175:     1181-1194.; Griendling K K, Minieri C A, Ollerenshaw J D & Alexander     R W (1994). Angiotensin II stimulates NADH and NADPH oxidase     activity in cultured vascular smooth muscle cells. Circulation     Research, 74:1141-1148.; Rajagopalan S, Surz S, Munzel T, Tarpey M,     Freeman B A, Griendling K K & Harrison D G (1996). Angiotensin     II-mediated hypertension in the rat increases vascular superoxide     production via membrane NADH/NADPH oxidase activation. Contribution     to alterations of vasomotor tone. Journal of Clinical Investigation,     97:1916-1923.; Schubert D, Behl C, Lesley R, Brack A, Dargusch R,     Sagara Y & Kimura H (1995). Amyloid peptides are toxic via a common     oxidative mechanism. Proceedings of the National Academy of     Sciences, USA, 92: 1989-1993.; Heinecke J W & Shapiro B M (1989).     Respiratory burst oxidase of fertilization. Proceedings of the     National Academy of Sciences, USA, 86: 1259-1263.; Aitken R J &     Clarkson J S (1987). Cellular basis of defective sperm function and     its association with the genesis of reactive oxygen species by human     spermatozoa. Journal of Reproduction and Fertility, 81: 459-469.; De     Lamirande E, Eiley D & Gagnon C (1993). Inverse relationship between     the induction of human sperm capacitation and spontaneous acrosome     reaction by various biological fluids and the superoxide scavenging     capacity of these fluids. International Journal of Andrology, 16:     258-266.; Acker H, Bolling B, Delpiano M A, Dufau E, Gorlach A &     Holtermann G (1992). The meaning of H₂O₂ generation in carotid body     cells for pO₂ chemoreception. Journal of the Autonomic Nervous     System, 41:41-51.; Cross A R, Henderson L, Jones O T G, Delpiano M     A, Hentschel J & Acker H (1990). Involvement of an NAD(P)H oxidase     as a pO₂ sensor protein in the rat carotid body. Biochemical     Journal, 272: 743-747.; Kummer W & Acker H (1995).     Immunohistochemical demonstration of four subunits of neutrophil     NAD(P)H oxidase in type I cells of carotid body. Journal of Applied     Physiology, 78: 1904-1909.; Schreck R, Rieber P & Baeuerle P A     (1991). Reactive oxygen intermediates as apparently widely used     messengers in the activation of the NFκB transcription factor and     HIV-1. EMBO Journal, 10: 2247-2258.; Menon S D, Quin S, Guy G R &     Tan Y H (1993). Differential induction of nuclear NFκB by protein     phosphatase inhibitors in primary and transformed human cells.     Requirement for both oxidation and phosphorylation in nuclear     translocation. Journal of Biological Chemistry, 268: 26805-26812.;     Baeuerle P A & Henkel T (1994). Function and activation of NM in the     immune system. Annual Review of Immunology, 12: 141-179.; Puri P L,     Avantaggiati M L, Burgio V L, Chirillo P, Collepardo D, Natoli G,     Balsano C & Levrero M (1995). Reactive oxygen intermediates mediate     angiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity     and proliferative hypertrophic responses in myogenic cells. Journal     of Biological Chemistry, 270: 22129-22134.; Park S J & Gunsalus R P     (1995). Oxygen, iron, carbon, and superoxide control of the fumarase     fumA and fumC genes of Escherichia coli: Role of the arcA, fnr, and     soxR gene products. Journal of Bacteriology, 177: 6255-6262.;     Hidalgo E & Demple B (1996). Activation of SoxR-dependent     transcription in vitro by noncatalytic or NifS-mediated assembly of     [2Fe-2S] clusters in Apo-SoxR. Journal of Biological Chemistry, 271:     7269-7272.; Jair K W, Fawcett W P, Fujita N, Ishihama A & Wolf Jr R     E (1996). Ambidextrous transcriptional activation by SoxS:     Requirement for the C-terminal domain of the RNA polymerase alpha     subunit in a subset of Escherichia coli superoxide-inducible genes.     Molecular Microbiology, 19: 307-317.; Christman M F, Storz G & Ames     B N (1989). OxyR, a positive regulator of hydrogen     peroxide-inducible genes in Escherichia coli and Salmonella     typhimurium, is homologous to a family of bacterial regulatory     proteins. Proceedings of the National Academy of Sciences, USA, 86:     3484-3488.; Marin-Hincapie M & Garofalo R S (1995). Drosophila     insulin receptor: lectin-binding properties and a role for     oxidative-reduction of receptor thiols in activation. Endocrinology,     136: 2357-2366.; Pan Z H, Bahring R, Grantyn R & Lipton S A (1995).     Differential modulation by sulfhydryl redox agents and glutathione     of GABA- and glycine-evoked currents in rat retinal ganglion cells.     Journal of Neuroscience, 15: 1384-1391.; Staal F J T, Anderson M T,     Staal G E J, Herzenberg L A & Gitler C (1994). Redox regulation of     signal transduction: Tyrosine phosphorylation and calcium influx.     Proceedings of the National Academy of Sciences, USA, 91:     3619-3622.; Hidalgo E, Bollinger Jr J M, Bradley T M, Walsh C T &     Demple B (1995). Binuclear [2Fe-2S] clusters in the Escherichia coli     SoxR protein and role of the metal centers in transcription. Journal     of Biological Chemistry, 270: 20908-20914.; Flint D H, Tuminello J F     & Emptage M H (1993). The inactivation of Fe—S cluster containing     hydrolases by superoxide. Journal of Biological Chemistry, 268:     22369-22376.; Halliwell B (1992). Switches in enzymes. Nature, 354:     191-192.; Bandyopadhyay S & Gronostajski R M (1994). Identification     of a conserved oxidation-sensitive cysteine residue in the NF1     family of DNA-binding proteins. Journal of Biological Chemistry,     269: 29949-29955.; Landgraf W, Regulla S, Meyer H E & Hofmann F     (1991). Oxidation of cysteines activates cGMP-dependent protein     kinase. Journal of Biological Chemistry, 266: 16305-16311.; Hayashi     T, Ueno Y & Okamoto T (1993). Oxidoreductive regulation of nuclear     factor κB: Involvement of a cellular reducing catalyst thioredoxin.     Journal of Biological Chemistry, 268: 11380-11388.; Petronilli V,     Constantini P, Scorrano L, Colonna R, Passamonti S & Bernardi P     (1994). The voltage sensor of the mitochondrial permeability     transition pore is tuned by the oxidation-reduction state of vicinal     thiols. Journal of Biological Chemistry, 269: 16638-16642.; Weiss S     J, Peppin G, Ortiz X, Ragsdale C & Test S T (1985). Oxidative     autoactivation of latent collagenase by human neutrophils. Science,     227: 747-749.; Weiss S J & Peppin G J (1986). Collagenolytic     metalloenzymes of the human neutrophil. Characteristics, regulation     and potential function in vivo. Biochemical Pharmacology, 35:     3189-3197.; Lander H M, Ogiste J S, Teng K K & Novogrodsky A (1995).     p21ras as a common signaling target of reactive free radicals and     cellular redox stress. Journal of Biological Chemistry, 270:     21195-21198.; Fialkow L, Chan C K, Grinstein S & Downey G P (1993).     Regulation of tyrosine phosphorylation in neutrophils by the NADPH     oxidase. Role of reactive oxygen intermediates. Journal of     Biological Chemistry, 268: 17131-17137.; Hardwick J S & Sefton M B     (1995). Activation of the Lck tyrosine protein kinase by hydrogen     peroxide requires the phosphorylation of Tyr-394. Proceedings of the     National Academy of Sciences, USA, 92: 4527-4531.; Ziegler D M     (1985). Role of reversible oxidation-reduction of enzyme     thiols-disulfides in metabolic regulation. Annual Review of     Biochemistry, 54: 305-329.; Korge P & Campbell K B (1993). The     effect of changes in iron redox state on the activity of enzymes     sensitive to modification of S H groups. Archives of Biochemistry     and Biophysics, 304: 420-428.; Johnson B D, Mancini-Samuelson G J &     Stankovich M T (1995). Effect of transition-state analogues on the     redox properties of medium-chain acyl-CoA dehydrogenase.     Biochemistry, 34: 7047-7055.; Hassoun P M, Yu F S, Zulueta J J,     White A C & Lanzillo J J (1995). Effect of nitric oxide and cell     redox status on the regulation of endothelial cell xanthine     dehydrogenase. American Journal of Physiology, 268: L809-L817.; Li     D, Stevens F J, Schiffer M & Anderson L E (1994). Mechanism of light     modulation: Identification of potential redox-sensitive cysteines     distal to catalytic site in light-activated chloroplast enzymes.     Biophysical Journal, 67: 29-35.; Drincovich M F & Andreo C S (1994).     Redox regulation of maize NADP-malic enzyme by thiol-disulfide     interchange: effect of reduced thioredoxin on activity. Biochimica     et Biophysica Acta, 1206: 10-16.; Terada T, Nanjo H, Shinagawa K,     Umemura T, Nishinaka T, Mizoguchi T & Nishihara T (1993). Modulation     of 3 ?-hydroxysteroid dehydrogenase activity by the redox state of     glutathione. Journal of Enzyme Inhibition, 7: 33-41.; Wunderlich M,     Jaenicke R & Glockshuber R (1993). The redox properties of protein     disulfide isomerase (DsbA) of Escherichia coli result from a tense     conformation of its oxidized form. Journal of Molecular Biology,     233: 559-566.; Wang G L, Jiang B H & Semenza G L (1995). Effect of     altered redox states on expression and DNA-binding activity of     hypoxia-inducible factor 1. Biochemical and Biophysical Research     Communications, 212: 550-556.; Arnone M I, Zannini M & Di Lauro R     (1995). The DNA binding activity and the dimerization ability of the     thyroid transcription factor I are redox regulated. Journal of     Biological Chemistry, 270: 12048-12055.; Esposito F, Cuccovillo F,     Morra F, Russo T & Cimino F (1995). DNA binding activity of the     glucocorticoid receptor is sensitive to redox changes in intact     cells. Biochimica et Biophysica Acta, 1260: 308-314.; Ammendola R,     Mesuraca M, Russo T & Cimino F (1994). The DNA-binding efficiency of     Sp1 is affected by redox changes. European Journal of Biochemistry,     225: 483-489.; Gozlan H, Khazipov R & Ben-Ari Y (1995). Multiple     forms of long-term potentiation and multiple regulatory sites of     N-methyl-D-aspartate receptors: role of the redox site. Journal of     Neurobiology, 26: 360-369.; Sullivan J M, Traynelis S F, Chen H S,     Escobar W, Heinemann S F & Lipton S A (1994). Identification of two     cysteine residues that are required for redox modulation of NMDA     subtype of glutamate receptor. Neuron, 13: 929-936.; Tang L H &     Aizenman E (1993). Long-lasting modification of the     N-methyl-D-aspartate receptor channel by a voltage-dependent     sulfhydryl redox process. Molecular Pharmacology, 44: 473-478.; Liu     G & Pessah I N (1994). Molecular interaction between ryanodine     receptor and glycoprotein triadin involves redox cycling of     functionally important hyperreactive sulfhydryls. Journal of     Biological Chemistry, 269: 33028-33034.; Galang C K & Hauser C A     (1993). Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein     is under redox regulation in vitro. Molecular and Cellular Biology,     13: 4609-4617.; Myrset A H, Bostad A, Jamin N, Lirsac P N, Toma F &     Gabrielsen O S (1993). DNA and redox state induced conformational     changes in the DNA-binding domain of the Myb oncoprotein. EMBO     Journal, 12: 4625-4633.; Hainaut P & Milner J (1993). Redox     modulation of p53 conformation and sequence-specific DNA binding in     vitro. Cancer Research, 53: 4469-4473.; Sanchez-Garcia I & Rabbitts     T H (1993). Redox regulation of in vitro DNA-binding activity by the     homeodomain of the Isl-1 protein. Journal of Molecular Biology, 231:     945-949.; Rondon I J, Scandurro A B, Wilson R B & Beckman B S     (1995). Changes in redox affect the activity of erythropoietin RNA     binding protein. FEBS Letters, 359: 267-270.; Michael Story and     Reinhard Kodym, Signal Transduction During Apoptosis; Implications     For Cancer Therapy, Frontiers in Bioscience, 3, d365-375, (Mar. 23,     1998).

Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H₂O₂ may act as signal transduction messengers. At least two well-defined transcription factors, nuclear factor (NM) and activator protein (AP)-1 have been identified to be regulated by the intracellular redox state. R. Schreck, P. Rieber & P. A. Baeuerle, Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1. EMBO J 10: 2247-2258 (1991). Binding sites of the redox-regulated transcription factors NFκB and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g., AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems may therefore be considered to be effective regulators of redox-sensitive gene expression. By controlling redox cascades by using antioxidants, for example, treatments for several diseases may be possible, such as hematogenic cancer cell metastasis and AIDS. See, Sen, C. K., Packer, L. Antioxidant and redox regulation of gene transcription. FASEB J. 10, 709-720 (1996). See, also: Packer L, Roy S, Sen C K. a-Lipoic acid: a metabolic antioxidant and potential redox modulator of transcription Advances in Pharmacology 1996; 38: 79-101.; Sen, C. K., S. Roy, and L. Packer. Therapeutic potential of the antioxidant and redox properties of a-lipoic acid. In: Oxidative Stress, Cancer, AIDS and Neurodegenerative Diseases. Eds. L. Montagnier, R. Olivier, C. Pasquier. Marcel Dekker Inc., New York, in press.; Packer L, Witt E H, Tritschler H J. Alpha-lipoic acid as a biological antioxidant. Free Rad. Biol. Med. 1995; 19: 227-250.; Sen C K, Atalay M, Hanninen O. Exercise-induced oxidative stress: glutathione supplementation and deficiency. J. Appl. Physiol. 1994; 77: 2177-2187.; Roy S, Sen C K, Tritschler H J, Packer L. Modulation of cellular reducing equivalent homeostasis by a-lipoic acid: mechanisms and implications for diabetes and ischemic injury. Biochem. Pharmacol., in press, 1996.; Arne E S J, Nordberg J, Holmgren A. Efficient reduction of lipoamide and lipoic acid by mammalian thiredoxin reductase. Biochem. Biophys. Res. Commun. 1996 in press.; Rosenberg H R, Culik R. Effect of a-lipoic acid on vitamin C and vitamin E deficiencies. Arch. Biochem. Biophys 1959; 80:86-93.; Baeuerle P A, Henkel T. Function and activation of NFκB in immune system. Annu. Rev. Immunol. 1994; 12: 141-179.; Staal F J T, Roederer M, Herzenberg L A, Herzenberg L A. Intracellular thiols regulate activation of nuclear factor κB and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 1990; 87: 9943-9947.; Sen C K, Roy S, Packer L. Involvement of intracellular Ca2+ in oxidant-induced NFκB activation. FEBS Letters 1996; 385: 58-62.; Watt F, Molloy P L. Specific cleavage of transcription factors by thiol protease, m-calpain. Nucleic Acid Res. 1993; 21: 5092-5100.; Yan C H I, Ferrari G, Greene L A. N-Acetylcysteine-promoted survival of PC12 cells is glutathione-independent but transcription-dependent. J. Biol. Chem. 1995; 270: 26827-26832; Baur A, Harrer T, Peukert M, Jahn G, Kalden J R, Fleckenstein B. Alpha-lipoic acid is an effective inhibitor of human immuno-deficiency virus (HIV-1) replication. Klin. Wochenschr. 1991; 69: 722-724.; Papp B, Bryn R A. Stimulation of HIV expression by intracellular calcium pump inhibition. J. Biol. Chem. 1995; 270: 10275-10283.; Eck H P, Gmunder H, Hartmann M, Petzoldt D, Daniel V, Droge W. Low concentrations of acid soluble thiol (cysteine) in blood plasma of HIV-1 infected patients. Biol. Chem. Hoppe-Seyler 1989; 370:101-108.; Droege W, Eck H-P, Naher H, Pekar U, Daniel V. Abnormal amino-acid concentrations in blood of patients with acquired immunodeficiency syndrome (AIDS) may contribute to the immunological defect. Biol. Chem. Hoppe. Seyler 1988; 369: 143-148.; Droege W, Eck H-P, Mihm S. HIV-induced cysteine deficiency and T cell dysfunction—a rationale for treatment with N-acetylcysteine. Immunol. Today. 1992; 13: 211-214.; Roederer M, Staal F J T, Anderson M E, Rabin R, Raju P A, Herzenberg L. A, Herzenberg L A. Disregulation of leukocyte glutathione in AIDS. Ann. NY Acad. Sci. 1993; 677:113-125.

-   20. Herzenberg L et al. In: Oxidative Stress, Cancer, AIDS and     Neurodegenerative Diseases. Eds. L. Montagnier, R. Olivier, C.     Pasquier. Marcel Dekker Inc., New York, in press.; Merin J P,     Matsuyama M, Kira T, Baba M, Okamoto T. a-Lipoic acid blocks HIV-1     LTR-dependent expression of hygromycin resistance in THP-1 stable     transforms. FEBS Letters; 1996, in press.

The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. H₂O₂ has been shown to induce a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H₂O₂ than with HS, thus providing evidence of a dual regulation of HSF by oxidants. The effects of H₂O₂ in vitro were reversed by the sulfhydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX). In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H₂O₂. Thus, H₂O₂ exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H₂O₂ favors the nuclear translocation of HSF, while on the other hand, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. Muriel R. Jacquier-Sarlin and Barbara S. Polla, “Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H₂O₂: role of thioredoxin”. (1996).

The mammalian stress response evokes a series of neuroendocrine responses that activate the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Coordinated interactions between stress response systems, occurring at multiple levels including the brain, pituitary gland, adrenal gland, and peripheral tissues, are required for the maintenance of homeostatic plateau. Glucocorticoids, as a major peripheral effector of the HPA axis, play an essential role in re-establishing homeostatic status in every peripheral tissue in human. On the other hand, the adaptive responses are also operated against various intrinsic or extrinsic forces that disturb cellular homeostasis as a part of local host-defense mechanisms at a cellular level. Currently, reduction/oxidation (redox) reactions are intimately involved in the control of biological processes including modulation of the function of transcription factors, e.g., AP-1 and NFκB. Cells contain endogenous buffering systems against excessive production of reactive oxygen intermediates (ROIs) to preserve cellular metabolism through the expression and regulation of many enzymes.

Glucocorticoids, on binding to the glucocorticoid receptor (GR), promote the dissociation of heat shock proteins (HSPs), and the ligand-receptor complex translocates to the nucleus then binds to palindromic DNA sequences, called glucocorticoid response elements (GREs). After binding to DNA, the GR differentially regulates target gene expression to produce hormone action, interacting with or without other transcription factors and coactivators/corepressors. The GR has a modular structure mainly consisting of a central DNA binding domain (DBD), nuclear localization signals, a ligand binding domain (LBD), and several transcription activation functions. The human GR contains 20 cysteine residues, concentrated in the central region spanning the DBD and LBD. The cysteine residues in each domain have been shown to be crucial for maintaining both structure and function of those domains. For examples, it has already been shown that conversion of sulfhydryls in the DBD to disulfides blocks GR binding to DNA cellulose, and that metal ions, which have high affinity for thiols interfere with the DBD-DNA interaction.

The TRX system operates as an endogenous defense machinery for glucocorticoid-mediated stress responses against oxidative stress. TRX is considered to be involved in transcriptional processes: for example, NFκB activation is inhibited, whereas AP-1 activity is induced by TRX. Moreover, the GR in the isolated rat cytosol is shown to be stabilized and maintained in their reduced, ligand-binding form by TRX. The functional interaction between cellular oxistress, TRX, and GR, and indicate that cellular redox state and TRX levels are important determinants of cellular sensitivity to glucocorticoids. Thus, TRX systems may control homeostasis not only by, for example, sequestrating ROIs, but also by fine tuning of hormonal signals. These phenomena appear to be rationale, for example during inflammation, where cells are believed to be exposed to severe oxidative stress, where suppression of glucocorticoid action may potentiate endogenous defense mechanisms and prevent premature termination of the cascade of inflammatory reactions for self defense. Increase in cellular TRX levels may restore the receptor activity and permit the GR to efficiently communicate with target genes. Resultant activation of anti-inflammatory genes and/or repression of inflammatory genes may prevent overshoot of inflammation. This process may be modulated by an alteration of the redox potential of the cell and the concentration of reduced GSH in the intracellular fluid. Yuichi Makino, Kensaku Okamoto, Kiichi Hirota, Junji Yodoi, Kazuhiko Umesono, Isao Makino, and Hirotoshi Tanaka, Cross-Talk between Endocrine Control of Stress Response and Cellular Antioxidant Defense System, Thioredoxin is a Redox-Regulating Cellular Cofactor for Glucocorticoid Hormone Action (Poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996 Dec. 9-20 Riken, Tsukuba, Japan. Therefore, glucocorticoid function may be modulated by glutathione administration. Thus, treatment of chronic inflammatory conditions, such as rheumatoid arthritis, as well as other immune and autoimmune disorders, may also benefit from treatment with glutathione. See: Kuehl, F. A., Ham, E. A., Egan, R. W., Dougherty, H. W., Bonney, R. J. and Humes, J. L.: Studies on a destructive oxidant released in the enzymatic reduction of prostaglandin G2 and other hydroperoxy acids. In: Pathology of Oxygen, ed. A. P. Auton, Acad. Press, New York, 1982, pp. 175-190.; Lash, L. H., Hagen, T. M., & Jones, D. P.: Exogenous glutathione protects intestinal epithelial cells from oxidative injury. Proc. Natl. Acad. Sci. USA 83:4641-4645, 1986.; Selye, H. 1946. The general adaptation syndrome and the diseases of adaptation. J. Clin. Endocrinol. Metab. 6:117-230.; Munck, A., P. M. Guyre, and N. J. Holbrook. 1984. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrine Rev. 5:25-44.; Yu, B. P. 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74:139-162.; Bauskin, A. R., I. Aikalay, and Y. Ben-Neriah. 1991. Redox regulation of a protein tyrosine kinase in the endoplasmic reticulum. Cell 66:685-696.; Demple, B., and C. F. Amabile-Cuevas. 1991. Redox redux: the control of oxidative stress responses. Cell 67:837-839.; Firth, J. D., B. L. Ebert, C. W. Pugh, and P. J. Ratcliffe. 1994. Oxygen-regulated control elements in the phosphoglycerate kinase and lactate dehydrogenase A genes: similarities with the erythropoietin 3′ enhancer. Proc. Natl. Acad. Sci. USA. 91:6496-6500.; Devary, Y., R. A. Gottlieb, T. Smeal, and M. Karin. 1992. The mammalian ultraviolet is triggered by activation of Src tyrosine kinases. Cell 71: 1081-1091.; Schreck, R, P. Rieber, and P. A. Baeuerle. 1991. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NFκB transcription factor and HIV-1. EMBO J. 10:2247-2258.; Abate, C., L. Patel, F. J. Rauscher, III, and T. Curran. 1990. Redox regulation of Fos and Jun DNA-binding activity in vitro. Science 249:1157-1161.; Klebanoff, S. J., M. A. Vadas, J. M. Harlan, L. H. Sparks, J. R. Gamble, J. M. Agosti, and A. M. Waltersdorf. 1986. Stimulation of neutrophils by tumor necrosis factor. J. Immunol. 136, 4220-4225.; Yoshie, O., T. Majima, and H. Saito. 1989. Membrane oxidative metabolism of human eosinophilic cell line EoL-1 in response to phorbol diester and formyl peptide: synergistic augmentation by interferon-gamma and tumor necrosis factor. J. Leukocyte Biol. 45, 10-20.; DeChatelet, L. R., P. S. Shirley, and R. B. Johnston. 1976. Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes. Blood 47, 545-554.; Beato M, P. Herrlich, and G. Schütz. 1995. Steroid receptors: many actors in search of a plot. Cell 83:851-857.; Evans, R. M. 1988. The steroid and thyroid hormone receptor superfamily. Science 240: 889-895.; Glass, K. C. 1994. Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Mol. Endocrinol. 15:391-407.; Hörlein, A. J., A. M. Näár, T. Heinzel, J. Torchis, B. Gloss, R. Kurokawa, A. Ryan, Y. Kamei, M. Söderström, C. K. Glass, and M. G. Rosenfeld. 1995. Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397-404.; Katzenellenbogen, J. A., B. W. O'Malley, and B. S. Katzellenbogen. 1996. Tripartite steroid hormone receptor pharmacology: interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Mol. Endocrinol. 10:119-131.; Onate, S. A., S. Y. Tsai, M.-J., Tsai, and B. W. O'Malley. 1995. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354-1357.; Kamei, Y., L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.-C. Lin, R. A. Heyman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1996. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403-414.; Chakraborti, P. K., M. J. Garabedian, K. R. Yamamoto, and S. S. Simons, Jr. 1992. Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid. J. Biol. Chem. 267:11366-11373.; Simons, S. S. Jr., and W. B. Pratt. 1995. Glucocorticoid receptor thiols and steroid-binding activity. Methods Enzymol. 251:406-422.; Luisi, B. F., W. X. Xu, Z. Otwinowski, L. P. Freedman, K. R. Yamamoto, and P. B. Sigler. 1991. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497-505.; Bodwell, J. E., N. J. Holbrook, and A. Munck. 1984. Sulfhydryl-modifying reagents reversibly inhibit binding of glucocorticoid-receptor complexes to DNA-cellulose. Biochemistry 23:1392-1398.; Makino Y., H. Tanaka, K. Dahlman-Wright, and I. Makino. 1996. Modulation of glucocorticoid-inducible gene expression by metal ions. Mol. Pharmacol. 49:612-620.; Holmgren, A. 1995. Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3:239-243.

-   26. Holmgren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54:237-271.;     Tagaya, Y., Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N.     Brown, K.-I. Arai, T. Yokota, H. Wakasugi, and J. Yodoi. 1989.     ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to     thioredoxin; possible involvement of dithiol-reduction in the IL-2     receptor induction. EMBO J. 8:757-764.; Tagaya, Y., M. Okada, K.     Sugie, T. Kasahara, N. Kondo, J. Hamuro, K. Matsushima, C. A.     Dinarello, and J. Yodoi. 1988. IL-2 receptor (p55)/Tac-inducing     factor. Purification and characterization of adult T cell     leukemia-derived factor. J. Immunol. 140:2614-2620.; Wakasugi,     N., Y. Tagaya, H. Wakasugi, A. Mitsui, M. Maeda, J. Yodoi, and T.     Tursz. 1990. Adult T-cell leukemia-derived factor/tyhioredoxin,     produced by both human T-lymphotrophic virus type I- and     Epstein-Barr virus-transformed lymphocytes, acts as an autocrine     growth factor and synergizes with interleukin 1 and interleukin 2.     Proc. Natl. Acad. Sci. USA. 87:8282-8286.; Schenk, H., M. Klein, W.     Erdbrugger, W. Droge, and K. Schulze-Osthoff. 1994. Distinct effects     of thioredoxin and antioxidants on the activation of transcription     factors NFκB and AP-1. Proc. Natl. Acad. Sci. USA. 91:1672-1676.;     Meyer, M., R. Schreck, and P. A. Baeuerle. 1993. H2O2 and     antioxidants have opposite effects on activation of NFκB and AP-1 in     intact cells: AP-1 as secondary antioxidant-responsive factor.     EMBO J. 12:2005-2015.; Grippo, J. F., A. Holmgren, and W. B.     Pratt. 1985. Proof that the endogenous, heat-stable glucocorticoid     receptor-activating factor is thioredoxin. J. Biol. Chem.     260:93-97.; Makino, Y., K. Okamoto, N. Yoshikawa, M. Aoshima, K.     Hirota, J. Yodoi, K. Umesono, I. Makino, and H. Tanaka. Thioredoxin:     a Redox-Regulating Cellular Cofactor for Glucocorticoid Hormone     Action. J. Clin. Invest. (in press); Sasada, T., S. Iwata, N.     Sato, Y. Kitaoka, K. Hirota, K. Nakamura, A. Nishiyama, Y.     Taniguchi, A. Takabayashi, and J. Yodoi. 1996. Redox control of     resistence to cis-diamminedichloroplatinum (II) (CDDP). Protective     effect of human thioredoxin against CDDP-induced cytotoxicity. J.     Clin. Invest. 97:2268-2276.; Alksnis, M., T. Barkhem, P.-E.     Stromstedt, H. Ahola, E. Kutoh, J.-Å. Gustafsson, L. Poellinger,     and S. Nilson. 1991. High level expression of functional full length     and truncated glucocorticoid receptor in Chinese hamster ovary     cells. J. Biol. Chem. 266:10078-10085.; Tagaya, Y., H. Wakasugi, H.     Masutani, H. Nakamura, S. Iwata, A. Mitsui, S. Fujii, N.     Wakasugi, T. Tursz, and J. Yodoi. 1990. Role of ATL-derived factor     (ADF) in the normal and abnormal cellular activation: involvement of     dithiol related reduction. Mol. Immunol. 27:1279-1289.;     Rangarajan, P. N., K. Umesono, and R. M. Evans. 1992. Modulation of     glucocorticoid receptor function by protein kinase A. Mol.     Endocrinol. 6:1451-1457.; Matthews, J. R., N. Wakasugi, J.-L.     Virelizer, J. Yodoi, and R. T. Hay. 1992. Thioredoxin regulates the     DNA binding activity of NFκB by reduction of a disulphide bond     involving cysteine 62. Nucleic Acids Res. 20:3821-3830.; Yokomizo,     A., M. Ono, H. Nanri, Y. Makino, T. Ohga, M. Wada, T. Okamoto, J.     Yodoi, M. Kuwano, and K. Kohno. 1995. Cellular levels of thioredoxin     associated with drug sensitivity to cisplatin, mitomycin C,     doxorubicin, and etoposide. Cancer Res. 55:4293-4296.; Makino,     Y., H. Tanaka, and I. Makino. 1994. Paradoxical derepression of the     collagenase gene expression by the anti-rheumatic gold compound     aurothiomalate. Mol. Pharmacol. 46:1084-1089.; Tanaka, H., Y.     Makino, K.-D. Wright, J.-A. Gustafsson, K. Okamoto, and I.     Makino. 1995. Zinc ions antagonize the inhibitory effect of     aurothiomalate on glucocorticoid receptor function at physiological     concentrations. Mol. Pharmacol. 48:938-945.; Sachi, Y., K.     Hirota, H. Masutani, K. Toda, T. Okamoto, M. Takigawa, and J.     Yodoi. 1995. Induction of ADF/TRX by oxidative stress in     keratinocytes and lymphoid cells. Immunol. Lett. 44, 189-193.;     Cappel, R. E., and H. F. Gilbert. 1988. Thiol/disulfide exchange     between 3-hydroxy-3-methyglutaryl-CoA reductase and glutathione. J.     Biol. Chem. 263:12201-12212.; Snyder, G. H., M. J. Cennerazzo, A. J.     Karalis, and D. Field. 1981. Electrostatic influence of local     cysteine environments on disulfide exchange kinetics. Biochemistry     20:6509-6518.; Xanthoudakis, S., G. Miao, F. Wang, Y.-C. E. Pan,     and T. Curran. 1992. Redox-activation of Fos-Jun DNA binding     activity is mediated by a DNA repair enzyme. EMBO J. 11: 3323-3335.;     Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schütz, K.     Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, and R. M.     Evans. 1995. The nuclear receptor superfamily: the second decade.     Cell 83:835-839.; Qin, J., G. M. Clore, W. M. P. Kennedy, J. R.     Huth, and A. M. Gronenborn. Solution structure of human thioredoxin     in a mixed disulfide intermediate complex with its target peptide     from the transcription factor NFκB. Structure 3:289-297.; Blake, M.     J., R. Udelsman, G. J. Feulner, D. D. Norton, and N. J.     Holbrook. 1991. Stress-induced heat shock protein 70 expression in     adrenal cortex: an adrenocorticotropic hormone-sensitive,     age-dependent response. Proc. Natl. Acad. Sci. USA. 88: 9873-9877.

The role of NFκB in HIV life cycle is critical especially in virus reactivation process within the latently infected cells has been widely accepted. After activation through intracellular signaling pathways such as those elicited by T cell receptor antigen complex or by receptors for IL-1 or TNF, NFκB initiates HIV gene expression by binding to the target DNA element within the promoter region of HIV LTR. Then, the virus-encoded trans-activator Tat is produced and triggers explosive viral replication. Since activation pathway of HIV gene expression by cellular transcription factor NFκB conceptually precedes activation by viral trans-activators, it is conceptual to ascribe NFκB as a determinant of the maintenance and breakdown of the viral latency. Antioxidants may be effective in treating AIDS by blocking HIV replication.

Another situation where NFκB plays a role is hematogenic cancer cell metastasis. NFκB induces E-selectin (also known as ELAM-1) on the surface of vascular endothelial cells. Since some cancer cells constitutively express a ligand for E-selectin, called sialyl-LewisX antigen, on their cell surface, induction of E-selectin is considered to be a rate determining step of cancer cell-endothelial cell interaction. For example, when primary human umbilical venous endothelial cells (HUVEC) are treated with IL-1 or TNF, nuclear translocation of NFκB is observed, followed by the augmented expression of E-selectin. In one study, the cell-to-cell interaction between HUVEC and QG90 cell, a tumor cell line derived from human small cell carcinoma of the lung expressing sialyl-LewisX antigen was studied, and it was found that IL-1 was able to induce the attachment of cancer cells to HUVEC. However, pretreatment of HUVEC with N-acetylcysteine, aspirin or pentoxyphillin efficiently blocked the cell-to-cell attachment in a dose-dependent manner. Okamoto. T. et al., Oxygen Radicals, Redox Regulation of the NFκB Signaling and Disease Control by Antioxidants (poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996 Dec. 9-20 Riken, Tsukuba, Japan. See, also: Ginn-Pease M E; Whisler R L, Redox signals and NFκB activation in T cells, Free Radic Biol Med. 1998 August; 25(3):346-61.; Holmgren A. Thioredoxin. Ann Rev Biochem 1985; 54: 237-271.; Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem 1989; 264, 13963-13966.; Ziegler D M. Role of reversible oxidation-reduction of enzyme thios-disulfides in metabolic regulation. Ann Rev Biochem 1985; 54, 305-329.; Allen J F. Redox control of transcription: sensors, response regulators, activators and repressors. FEBS Lett 1993; 332: 203-207.; Gilmore T D. NF-κB, KBF-1, dorsal and related matters. Cell 1990; 62: 841-843.; Baeuerle P A. The inducible transcription activator NF-κB: regulation by distinct protein subunits. Biochim Biophys Acta 1991; 1072: 63-80.; Baeuerle P A, Henkel T. Function and activation of NF-κB in the immune system. Ann Rev mmunol 1994; 12: 141-179.; Thanos D, Maniatis T. NF-κB: a lesson in family values. Cell 1995; 80: 529-532.; Schindler U, Baichwal V R. Three NF-κB binding sites in the human E-selectin gene required for maxmal tumor necrosis factor alpha-induced expression. Mol Cell Biol 1994; 14: 5820-5831.; Okamoto T, Matsuyama T, Mori S, Hamamoto Y, Kobayashi N, Yamamoto N, Josephs S F, Wong-Staal F, Shimotohno K. Augmentation of human immunodeficiency virus type 1 gene expression by tumor necrosis factor alpha. AIDS Res Hum Retrovir 1989; 5: 131-138.; Maekawa T, Itoh F, Okamoto T, Kurimoto M, Imamoto F, Shii S. Identification and purification of the enhancer-binding factor of human immunodeficiency virus-1. Multiple preteins and binding to other enhancers. J Biol Chem 1989; 264: 2826-2831.; Stade B G, Messer G, Riethmuller G., Johnson J P. Structural characteristics of the 5′ region of the human ICAM-1 gene. Immunobiol 1990; 182:79-87.; Mukaida N, Mahe Y, Matsushima K. Cooperative interaction of nuclear factor-κ B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem 1990; 265: 21128-21133.; Roebuck K A, Rahman A, Lakshminarayanan V, Janakidevi K, Malik A B. H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem 1995; 270:18966-18974.; Donnelly R P, Crofford L J, Freeman S L, Buras J, Remmers E, Wilder R L, Fenton M J. Tissue-specific regulation of IL-6 production by IL-4. Differential effects of IL-4 on nuclear factor-κ B activity in monocytes and fibroblasts. J mmunol 1993; 151: 5603-5612.; Schreck R, Baeuerle P A. NF-κB as inducible transcriptional activator of the granulocyte-macrophage colony-stimulating factor gene. Mol Cell Biol 1990; 10:12811286.; Staynov D Z, Cousins D J, Lee T H. A regulatory element in the promoter of the human granulocyte-macrophage colony-stimulating factor gene that has related sequences in other T-cell-expressed cytokine genes. Proc Natl Acad Sci USA 1995; 92: 3606-3610.; Xie Q-W, Kashiwabara Y, Nathan C. Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase. J Biol Chem 1994; 269: 4705-4708.; Sen R, Baltimore D. Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell 1986; 46: 705-716.; Nabel G, Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 1987; 326: 711-713.; Baeuerle P A, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor. Cell 1988A; 53: 211-217.; Baeuerle P A, Baltimore D. I-κB: a specific inhibitor of the NF-κB transcription factor. Science 1988B; 242: 540-546.; Ghosh S, Gifford A M, Riviere L R, Tempst P, Nolan G P, Baltimore D. Cloning of the p50 DNA binding subunit of NF-κB: homology to Rel and dorsal. Cell 1990; 62: 1019-1029.; Ghosh S, Baltimore D. Activation in vitro of NF-κB by phosphorylation of its inhibitor I-κB. Nature 1990; 344: 678-682.; Read M A, Whitley M Z, Williams A J, Collins T. The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. J Exp Med 1994; 179: 503-512.; Hayashi T, Sekine T, Okamoto T. Identification of a new serine kinase that activates NF-κB by direct phosphorylation. J Biol Chem 1993A; 826: 26790-26795.; Shirakawa F, Mizel S B. In vitro activation and nuclear translocation of NF-κB catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol 1989; 9: 2424-2430.; Meichle A, Schutze S, Hensel G, Brunsing D, Kronke M. Protein kinase C-independent activation of nuclear factor kB by tumor necrosis factor. J Biol Chem 1990; 265: 8339-8343.; Feuillard J, Gouy H, Bismuth G, Lee L M, Debre P, Korner M. Nf-κB activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca (2)-regulated kinase. Cytokine 1991; 3: 257-265.; Ostrowski J, Sims J E, Sibley C H, Valentine M A, Dower S K, Meier K E, Bomsztyk K. A serine/threonine kinase activity is clsely associated with a 65-kDa phosphoprotein specifically recognized by the κB enhancer element. J Biol Chem 1991; 266:12722-12733.; Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, Kronke M. TNF activates NF-κB by phosphatidylcholine-specific phospholipase C-induced “acidic” sphingomyelin breakdown. Cell 1992; 71: 765-776.; Brown K, Gerstberger S, Carlson L, Franzoso G, Sieben list U. Control of I-κ B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science 1995; 267:1485-1488.; Cao Z, Henzel W J, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 1996; 271:1128-1131.; Chen Z J, Parent L, Maniatis T. Site-specific phosphorylation of IkBa by a novel ubiquitination-dependent protein kinase activity. Cell 1996; 84: 853-862.; Naumann M, Scheidereit C. Activation of NF-κB in vivo is regulated by multiple phosphorylations. EMBO J 1994; 13: 4597-4607.; Li C-C H, Dai R-M, Chen E, Longo D L. Phosphorylation of NFKB1-p50 is involved in NF-κB activation and stable DNA binding. J Biol Chem 1994; 269: 30089-30092.

-   38. Okamoto T, Ogiwara H, Hayashi T, Mitsui A, Kawabe T, Yodoi J.     Human thioredoxin/adult T cell leukemia-derived factor activates the     enhancer binding protein of human immunodeficiency virus type 1 bt     thiol redox control mechanism. Int Immunol 1992; 4: 811-819.;     Hayashi T, Ueno Y, Okamoto T. Oxidoreductive regulation of nuclear     factor κB. Involvement of a cellular reducing catalyst thioredoxin.     J Biol Chem 1993B; 268: 11380-11388.; Tagaya Y, Maeda Y, Mitsui A,     Kondo N, Matsui H, Hamuro J, Brown J, Arai K I, Yokota T, Wakasugi     H, Yodoi J. ATL-derived factor (ADF), an IL-2 receptor/Tac inducer     homologous to thioredoxin; possible involvement of dithiol-reduction     in the IL-2 receptor induction. EMBO J 1989; 8: 757-764.; Schreck R,     Rieber P, Baeuerle P A. Reactive oxygen intermediates as apparently     widely used messengers in the activation of the NF-κB transcription     factor and HIV-1. EMBO J 1991; 10: 2247-2258.; Molitor J A, Ballard     D W, Greene W C. K-B-specific DNA binding proteins are     differentially inhibited by enhancer mutations and biological     oxidation. New Biol 1991; 3: 987-996.; Toledano M B, Leonard W J.     Modulation of transcription factor NF-κB binding activity by     oxidation-reduction in vitro. Proc Natl Acad Sci USA 1991; 88:     4328-4332. -   44. Matthews J R, Wakasugi N, Virelizier J-L, Yodoi J, Hay R T.     Thioredoxin regulates the DNA binding activity of NF-κB by reduction     of a disulfide bond involving cystein 62. Nucleic Acids Res 1992;     20, 3821-3830.; Ghosh G, Van Duyne G, Ghosh S, Sigler P B. Structure     of NF-κB p50 homodimer bound to a κB site. Nature 1995; 373:     303-310.; Muller C W, Rey F A, Sodeoka M, Verdine G L, Harrison S C.     Structure of the NF-κB p50 homodimer bound to DNA. Nature 1995; 373:     311-317.; Qin J, Clore G M, Kennedy W M P, Huth J R, Gronenborn A M.     Solution structure of human thioredoxin in a mixed disulfide     intermediate complex with its target peptide from the transcription     factor NF-κB. Structure 1995; 3: 289-297.; Roederer M, Staal F J T,     Raju P A, Ela S W, Herzenberg L A, Herzenberg L A.     Cytokine-stimulated human immunodeficiency virus replication is     inhibited by N-acetyl-Lcysteine. Proc Natl Acad Sci USA 1990; 87:     4884-4888.; Suzuki Y J, Aggarwal B B, Packer L. Alpha-lipoic acid is     a potent inhibitor if NF-κB activation in human T cells. Biochem     Biophys Res Commun 1992; 189: 1709-1715.; Meyer M, Schreck R,     Baeuerle P A. H2O2 and antioxidants have opposite effects on     activation of NF-κB and AP-1 in intact cells: AP-1 as secondary     antioxidant-responsive factor. EMBO J 1993; 12: 2005-2015.; Biswas D     K, Dezube B J, Ahlers C M, Pardee A B. Pentoxifylline inhibits HIV-1     LTR-driven gene expression by blocking NF-κB action. J AIDS 1993; 6:     778-786.; Suzuki Y J, Packer L. Signal transduction for nuclear     factor-κB activation. Proposed location of antioxidant-inhibitable     step. J Immunol 1994; 153: 5008-5015.; Packer L, Witt E H,     Tritschler H J. Alpha-lipoic acid as a biological antioxidant. Free     Rad Biol Med 1995; 19: 227-250.; Sachi Y, Hirota K, Masutani H, Toda     K, Okamoto T, Takigawa M, Yodoi J. Three NF-κB binding sites in the     human E-selectin gene required for maximal tumor necrosis factor     alpha-induced expression. Immunol Lett 1995; 44: 189-193.; Yang J P,     Merin J P, Nakano T, Kato T, Kitade Y, Okamoto T. Inhibition of the     DNA-binding activity of NF-κB by gold compounds in vitro. FEBS lett     1995; 361: 89-96.; Skosey J. L. in “Arthritis and allied conditions”     (McCarty D J, Koopman W J, eds) pp 603-614, Lea & Febiger,     Philadelphia, 1993.; Insel P A. in “Autacoids: Drug Therapy of     Inflammation” (Gilman G, et al, eds) pp. 670-681, Macmillan, New     York, 1990.; Handel M L, McMorrow L B, Gravallese E. M. Nuclear     factor-kB in rheumatoid synovium. Localization of p50 and p60.     Arthritis Rheum 1996; 38:1762-1770.; Sakurada S, Kato T, Okamoto T.     Induction of cytokines and ICAM-1 by proinflammatory cytokines in     primary rheumatoid synovial fibroblasts and inhibition by     N-acetyl-L-cysteine and aspirin. Int mmunol 1996 in press.; Bohnlein     E, Lowenthal J W, Siekevitz M, Ballard D W, Franza B R, Greene W C.     The same inducible nuclear proteins regulates mitogen activation of     both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell     1988; 53: 827-836.; Okamoto T, Benter T, Josephs S F, Sadaie M R,     Wong-Staal F. Transcriptional activation from the long-terminal     repeat of human immunodeficiency virus in vitro Virology 1990; 177:     606-614.; Arya S K, Guo C, Josephs S F, Wong-Staal F.     Trans-activator gene of human T-lymphotropic virus type III     (HTLV-III). Science 1985; 229: 69-73.; Sodroski J, Patarca R,     Rosen C. Location of the trans-activating region on the genome of     human T-cell lymphotropic virus type III. Science 1985; 229: 74-77.;     Okamoto T, Wong-Staal F. Demonstration of virus-specific     transcriptional activator(s) in cells infected with HTLV-III by an     in vitro cell-free system. Cell 1986; 47: 29-35.; Peterlin B M,     Luciw P A, Barr P J, Walker M D. Elevated levels of mRNA can account     for the trans-activation of human immunodeficiency virus. Proc Natl     Acad Sci USA 1986; 83: 9734-9738.; Tozawa K, Sakurada S, Kohri K,     Okamoto T. Effects of anti-nuclear factor κB reagents in blocking     adhesion of human cancer cells to vascular endothelial cells. Cancer     Res 1995; 55: 4162-4167.; Montgomery K F, Osborn L, Hession C,     Tizard R, Goff D, Vassallo C, Tarr P I, Bomsztyk K, Lobb R, Harlan J     M, Pohlman T H. Activation of endothelial-leukocyte adhesion     molecule 1 (ELAM-1) gene transcription. Proc Natl Acad Sci USA 1991;     88: 6523-6527.; Whelan J, Ghersa P, Huijsduijnen R H, Gray J,     Chandra G, Talabot F, DeLamarterJ F. An NFκB-like factor is     essential but not sufficient for cytokine induction of endothelial     leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Nuc Acid     Res 1991; 19: 2645-2653.; Dejana E, Bertocci F, Bortolami M C,     Regonesi A, Tonta A, Breviario F, Giavazzi R. Interleukin 1 promotes     tumor cell adhesion to cultured human endothelial cells. J Clin     nvest 1988; 82:1466-1470.; Takada A, Ohmori K, Yoneda T, Tsuyuoka K,     Hasegawa A, Kiso M, Kannagi R. Contribution of carbohydrate antigens     sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells     to vascular endothelium. Cancer Res 1993; 53: 354-361.; Kira T,     Merin J P, Baba M, Shigeta S, Okamoto T. Anti-Tat MTT assay: a novel     anti-HIV drug screening system using the viral regulatory network of     replication. AIDS Res Hum Retrovir 1995; 11: 1359-1366.

Pigmented Epithelium Derived Factor

It is well known that solid tumors, such as carcinomas, require neovascularization to continue growth beyond a few millimeters in size. This is because, as with all tissues, they need oxygen and must rid themselves of toxic metabolic products. Further, rapidly growing tumors may have demands well in excess of that of normal tissues due to a high rate of cell replication. Therefore, one technique which has been sought to be employed in fighting tumors is the use of pharmaceuticals and agents that block neovascularization, for example tumor necrosis factor, endostatin, angiostatin, and other agents. One agent that has aroused interest is Pigmented Epithelium Derived Factor (PEDF), a protein of the serine protease inhibitor (serpin) supergene family, but with characteristics of a substrate rather than inhibitor. PEDF was named for its association with the pigmented RPE cells of the macula, described above. See: Tombran-Tink et al., “Neuronal Differentiation of Retinoblastoma Cells Induced by Medium Conditioned by Human RPE Cells,” Investigative Ophthalmology & Visual Science, 30(8), 1700-1707 (1989); G Chader, SP Becerra, L Johnson, J Tombran-Tink, F Steele and I Rodriguez, PCT/US95/07201 filed 6 Jun. 95, published under WO 95/33480 on 14 Dec. 95; U.S. Ser. No. 07/952,796 entitled A DNA Clones for the Expression of Pigment Epithelium Derived Growth Factor and Related Proteins, filed Sep. 24, 1992 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink and Sofia P. Becerra; U.S. Ser. No. 08/257,963 entitled A Pigment Epithelium Derived Factor: Characterizations of Its Biological Activity and Sequences Encoding and Expressing the Protein, filed Jun. 7, 1994 by Gerald J. Chader, Sofia P. Becerra, Joan P. Schwartz, Takayuki Taniwaki and Yukihera Sugita, now U.S. Pat. No. 5,840,686; U.S. Ser. No. 08/279,979 entitled A Retinal Pigmented Epithelium Derived Neurotrophic Factor, filed Jul. 25, 1994 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra and Ignacio R. Rodriquez and Lincoln Johnson; U.S. Ser. No. 08/367,841 entitled A Pigment Epithelium Derived Factor: Characterization, Genomic Organization and Sequence of the PEDF Gene, filed Dec. 30, 1994 by Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra, Ignacio R. Rodriquez and Fintan R. Steele and Lincoln Johnson; U.S. Ser. No. 08/377,710 entitled A DNA Clones for the Expression of Pigment Epithelium Derived Factor and Related Proteins, filed Jan. 25, 1995 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra and Ignacio R. Rodriquez; U.S. Ser. No. 08/520,373 entitled A Retinal Pigmented Epithelium Derived Neurotrophic Factor, filed Aug. 29, 1995 by Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra, Ignacio R. Rodriquez and Fintan R. Steele; FR Steele, G J Chader, L V Johnson, J Tombran-Tink. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1526-1530; SP Becerra, I Palmer, A Kumar, F Steele, J Shiloach, V Notario, G J Chader. Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli: a functionally active neurotrophic factor. J Biol Chem 1993 Nov. 5; 268 (31): 23148-56; Perez-Mediavilla L A, Chew C, Campochiaro P A, Nickells R W, Notario V, Zack D J, Becerra S P, Sequence and expression analysis of bovine pigment epithelium-derived factor, Biochim Biophys Acta 1998 Jun. 16; 1398(2):203-14; Slavc I; Rodriguez I R; Mazuruk K; Chader G J; Biegel J A, Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors, Int J Cancer 1997; 72(2):277-82.

PEDF is a potent autocrine and paracrine hormone which blocks epithelial cell proliferation (including vascular epithelial cells, necessary for neovascularization), and promotes cellular differentiation, and is neurotrophic and neuroprotective. Sugita Y, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes, J Neurosci Res 1997 Sep. 15; 49(6):710-8. Subsequent studies have confirmed that PEDF or its isoforms are widely distributed throughout the body, but with relatively high concentration in the pigmented epithelial cells of the retina and central nervous system. PEDF may help cells resist apoptosis. Araki T, Taniwaki T, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death, J Neurosci Res 1998 Jul. 1; 53(1):7-15. Glutathione depletion has also been directly associated with failure of differentiation. Esposito F, Agosti V, Morrone G, Morra F, Cuomo C, Russo T, Venuta S, Cimino F, Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion, Biochem. J. (1994) 301, 649-653.

PEDF binds to extracellular matrixes. Alberdi E, Hyde C C, Becerra S P, Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site, Biochemistry 1998 Jul. 28; 37(30):10643-52 (Published erratum appears in Biochemistry 1998 Dec. 22; 37(51)18128).

PEDF is among the most potent direct angiogenesis factors known. In the eye, it prevents ingrowth of blood vessels in the lens, retina and vitreous body of the eye. Ortego J, Escribano J, Becerra S P, Coca-Prados M, Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor, Invest Ophthalmol Vis Sci 1996 December; 37(13):2759-67. PEDF is a dramatic enhancer of cellular differentiation, and is capable, for example, of inducing retinoblastoma cells to retrotransform into normal appearing cells. Stratikos E, Alberdi E, Gettins P G, Becerra S P, Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells, Protein Sci 1996 December; 5(12):2575-82. PEDF protects neural tissue against an array of injurious factors, for example, against the excitatory neurotoxicity of glutamate. Taniwaki T, Hirashima N, Becerra S P, Chader G J, Etcheberrigaray R, Schwartz J P, Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity, J Neurochem 1997 January; 68(1):26-32.

PEDF is produced by the stromal cells of the endometrium and has a strong effect on the growth and differentiation of the glandular epithelium. When stromal cells become deciduous cells, in response to hormones and pregnancy, PEDF production is considered crucial to prevent (i) uncontrolled growth and penetration of the otherwise highly invasive trophoblastic cells of the placenta, into the uterine wall, and (ii) uncontrolled ingrowth of the blood vessels from the chorionic villi, into the uterine wall.

PEDF controls the cell cycle in many different cell types, by a direct effect on cell cycle control factors. The source of PEDF, namely the retinal pigment epithelium (RPE), may be crucial to the normal development and function of the neural retina. A variety of biologically active molecules, including growth factors, are synthesized and secreted by RPE cells. The RPE develops prior to and lies adjacent to the neural retina, and that it functions as part of the blood-retina barrier. Fine et al., The Retina, Ocular Histology: A Text and Atlas, New York, Harper & Row, 61-70 (1979). the RPE has been implicated in vascular, inflammatory, degenerative, and dystrophic diseases of the eye. Einer et al., Am. J. Pathol., 136, 745-750 (1990). In addition to growth factors, nutrients and metabolites are also exchanged between the RPE and the retina. For example, the RPE supplies to the retina the well-known growth factors PDGF, FGF, TGFα, and TGFβ. Campochiaro et al., Invest. Ophthalmol. Vis. Sci., 29, 305-311 (1988); Plouet, Invest. Ophthalmol. Vis. Sci., 29, 106-114 (1988); Fassio et al., Invest. Ophthalmol. Vis. Sci., 29, 242-250 (1988); Connor et al., Invest. Ophthalmol. Vis. Sci., 29, 307-313 (1988). It is very likely that these and other unknown factors supplied by the RPE influence the organization, differentiation, and normal functioning of the retina.

In order to study and determine the effects of putative differentiation factors secreted by the RPE, cultured cells have been subjected to retinal extracts and conditioned medium obtained from cultures of human fetal RPE cells. For example, U.S. Pat. No. 4,996,159 (Glaser) discloses a neovascularization inhibitor recovered from RPE cells that is of a molecular weight of about 57,000±3,000. Similarly, U.S. Pat. No. 1,700,691 (Stuart), U.S. Pat. No. 4,477,435 (Courtois et al.), and U.S. Pat. No. 4,670,257 (Guedon born Saglier et al.) disclose retinal extracts and the use of these extracts for cellular regeneration and treatment of ocular disease. Furthermore, U.S. Pat. No. 4,770,877 (Jacobson) and U.S. Pat. No. 4,534,967 (Jacobson et al.) describe cell proliferation inhibitors purified from the posterior portion of bovine vitreous humor.

PEDF has been isolated from human RPE as a 50-kDa protein. Tombran-Tink et al., Invest. Ophthalmol. Vis. Sci., 29, 414 (1989); Tombran-Tink et al., Invest. Opthalmol. Vis. Sci., 30, 1700-1707 (1989); Tombran-Tink et al., “PEDF: A Pigment Epithelium-derived Factor with Potent Neuronal Differentiative Activity,” Experimental Eye Research, 53, 411-414 (1991). Specifically, PEDF has been demonstrated to induce the differentiation of human Y79 retinoblastoma cells, which are a neoplastic counterpart of normal retinoblasts. Chader, Cell Different., 20, 209-216 (1987); Taniwaki T, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture, J Neurochem 1995 June; 64(6):2509-17. The differentiative changes induced by PEDF include the extension of a complex meshwork of neurites, and expression of neuronal markers such as neuron-specific enolase and neurofilament proteins. This is why the synthesis and secretion of PEDF protein by the RPE is believed to influence the development and differentiation of the neural retina. Furthermore, PEDF is only highly expressed in undifferentiated human retinal cells, like Y79 retinoblastoma cells, but is either absent or down-regulated in their differentiated counterparts. It was also reported that PEDF mRNA is expressed in abundance in quiescent human fetal W1 fibroblast cells and not expressed in their senescent counterparts. Pignolo et al. (1993), J. Biol. Chem., 268:2949-295.

Further study of PEDF and examination of its potential therapeutic use in the treatment of inflammatory, vascular, degenerative, and dystrophic diseases of the retina and central nervous system (CNS) necessitates obtaining large quantities of PEDF. Unfortunately, the low abundance of PEDF in fetal human eye and, furthermore, the rare availability of its source tissue, especially in light of restrictions on the use of fetal tissue in research and therapeutic applications, make further study of PEDF difficult at best. Therefore, a recombinant technique was developed to procure a supply of the factor. See, U.S. Pat. No. 5,840,686, supra.

Based upon the protein amino acid sequence, PEDF has been found to have extensive sequence homology with the serpin gene family, members of which are serine protease inhibitors. Many members of this family have a strictly conserved domain at the carboxyl terminus which serves as the reactive site of the protein. These proteins are thus thought to be derived from a common ancestral gene. However the developmental regulation differs greatly among members of the serpin gene family and many have deviated from the classical protease inhibitory activity. Becerra S P, Structure-function studies on PEDF. A noninhibitory serpin with neurotrophic activity, Adv Exp Med Biol 1997; 425:223-37. Although PEDF shares sequence homology with serpins, analysis of the cDNA sequence indicates that it lacks the conserved domain and thus may not function as a classical protease inhibitor.

Genomic sequencing and analysis of PEDF has provided sequences of introns and exons as well as approx. 4 kb of 5′-upstream sequence. The gene for PEDF has been localized to 17p13.1 using both in situ hybridization and analyses of somatic cell hybrid panels. Tombran-Tink, et al., (1994) Genomics, 19:266-272. This is very close to the p53 tumor suppressor gene as well as to the chromosomal localization of a number of hereditary cancers unrelated to mutations in the p53 gene product PEDF thus becomes a prime candidate gene for these cancers.

Although PEDF is particularly highly expressed by RPE cells, it is detectable in most tissues, cell types, tumors, etc. by Northern and Western blot analyses. It is readily detected, for example in vitreous and aqueous humors. The important question of subcellular localization of PEDF has also been addressed. Although the bulk of the PEDF appears to be secreted, PEDF is also associated with the nucleus as well as with very specific cytoskeletal structures in the cytoplasm. Importantly, this varies as to the age of the cells and the specific cell-cycle state. For example, the protein appears to concentrate at the tips of the pseudopods of primate RPE cells that interact with the substratum during the initial stages of attachment. Later though, this staining disappears and there is appearance of the protein in association with specific cytoskeletal structures and the nucleus. Thus it appears that PEDF plays an important intracellular role in both nucleus and cytoplasm.

There is PEDF expression in dividing, undifferentiated Y-79 cells and little or no expression in their quiescent, differentiated counterparts. Tombran-Tink, et al., (1994) Genomics, 19:266-272. The synthesis of PEDF in WI-38 fibroblast cells is restricted to the G₀ stage of the cell cycle in young cells. Pignolo et al. (1993), J. Biol. Chem., 268:2949-295. Moreover, in old senescent cells, PEDF messenger RNA is absent.

In the retina, PEDF inhibits the Muller glial cells. Since Muller cells are similar to astroglia, PEDF would be similarly effective in blocking gliosis in conditions such as retinal detachment, diabetes, Retinitis Pigmentosa, etc. as well as sparing the lives of the retinal neurons. Thus, administration of glutathione, to alter cellular redox potential, and thereby alter PEDF expression, may have particular value.

Apparently, in macular degeneration, the pigmented RPE cells become defective, and die, resulting in a functional loss of PEDF in the macula. Without the continuous presence of PEDF, vascular epithelial cells undergo a de-differentiation and enter into a proliferative stage, resulting in neovascularization, with invasion of the cornea in vitreous with blood vessels. The amount of inhibitory PEDF produced by retinal cells is positively correlated with oxygen concentration. Thus, PEDF is presumed to play a role in ischemia-driven retinal neovascularization. In fact, studies have shown that it is not necessary to kill the RPE cells to reduce PEDF availability. The availability of PEDF is sensitive to the redox potential of the cell, being more available in a reduced state and less available when the cell is in an oxidized state. (Ischemia is associated with a state in which cells produce an excess of free radicals. These may be due to exhaustion of antioxidants, cell death or apoptosis, or accumulation of toxic metabolic waste). This feedback regulation, which is applicable to other PEDF producing cells, thus induces vascularization where blood flow is needed (relatively oxidized redox potential) while maintaining an appropriate balance and allowing certain privileged tissues to remain unvascularized or with highly controlled vascularization. The oxidative control over PEDF is believed to be at the translative or post-translative levels, as mRNA levels are generally unchanged. It is noted that other classes of biologically active agents respond to redox state through transcriptional modification or sensitivity.

Efforts to directly administer PEDF, a peptide hormone, are met with difficulty, due to both the unavailability of bulk quantities of PEDF and difficulties in administration thereof. See: Alberdi E, et al., Binding of Pigment Epithelium-derived Factor (PEDF) to Retinoblastoma Cells and Cerebellar Granule Neurons. Evidence for a pedf receptor. J Biol Chem. 1999 Oct. 29; 274(44):31605-31612.; Cao W, et al., Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. J. Neurosci Res. 1999 Sep. 15; 57(6):789-800.; Houenou L J, et al., Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons. J Comp Neurol. 1999 Sep. 27; 412(3):506-14.; Bilak M M, et al., Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J. Neuropathol Exp Neurol. 1999 July; 58(7):719-28.; Koenekoop R, et al., Four polymorphic variations in the PEDF gene identified during the mutation screening of patients with Leber congenital amaurosis., Mol Vis. 1999 Jul. 2; 5:10.; Dawson D W, et al., Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science. 1999 Jul. 9; 285(5425):245-8.; DeCoster M A, et al., Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons. J Neurosci Res. 1999 Jun. 15; 56(6):604-10.; Tresini M, et al., Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts. J Cell Physiol. 1999 April; 179(1):11-7.; Palmieri D, et al., Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence. Exp Cell Res. 1999 Feb. 25; 247(1)142-7.; Malchiodi-Albedi F, et al., PEDF (pigment epithelium-derived factor) promotes increase and maturation of pigment granules in pigment epithelial cells in neonatal albino rat retinal cultures. Int J Dev Neurosci. 1998 August; 16(5):423-32.; Alberdi E, et al., Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site. Biochemistry. 1998 Jul. 28; 37(30)10643-52.; Perez-Mediavilla L A, et al., Sequence and expression analysis of bovine pigment epithelium-derived factor. Biochim Biophys Acta. 1998 Jun. 16; 1398(2):203-14.; Araki T, et al., Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death. J Neurosci Res. 1998 Jul. 1; 53(1):7-15.; Carwile M E, et al., Rod outer segment maintenance is enhanced in the presence of bFGF, CNTF and GDNF. Exp Eye Res. 1998 June; 66(6):791-805.; Kozaki K, et al., Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells. J Biol Chem. 1998 Jun. 12; 273(24)15125-30.; Singh V K, et al., Structural and comparative analysis of the mouse gene for pigment epithelium-derived factor (PEDF). Mol Vis. 1998 Apr. 20; 4:7.; Broekhuyse R M, et al., Differential effect of macrophage depletion on two forms of experimental uveitis evoked by pigment epithelial membrane protein (EAPU), and by melanin-protein (EMIU). Exp Eye Res. 1997 December; 65(6):841-8.; Becerra S P, Structure-function studies on PEDF. A noninhibitory serpin with neurotrophic activity. Adv Exp Med Biol. 1997; 425:223-37. Review.; Sugita Y, et al., Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes. J Neurosci Res. 1997 Sep. 15; 49(6):710-8.; Slavc I, et al., Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors. Int J Cancer. 1997 Jul. 17; 72(2):277-82.; Taniwaki T, et al., Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. J Neurochem. 1997 January; 68(1):26-32.; Ortego J, et al., Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor. Invest Ophthalmol Vis Sci. 1996 December; 37(13):2759-67.; Stratikos E, et al., Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells. Protein Sci. 1996 December; 5(12):2575-82.; Tombran-Tink J, et al., Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin. Mol Vis. 1996 Nov. 4; 2:11.; Wu Y Q, et al., Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing. Invest Ophthalmol Vis Sci. 1996 September; 37(10):1984-93.; Goliath R, et al., The gene for PEDF, a retinal growth factor is a prime candidate for retinitis pigmentosa and is tightly linked to the RP13 locus on chromosome 17p13.3. Mol Vis. 1996 Jun. 19; 2:5.; Lotery A J, et al., Localisation of a gene for central areolar choroidal dystrophy to chromosome 17p. Hum Mol Genet. 1996 May; 5(5):705-8.; Phillips N J, et al., Allelic deletion on chromosome 17p13.3 in early ovarian cancer. Cancer Res. 1996 Feb. 1; 56(3):606-11.; Balciuniene J, et al., A gene for autosomal dominant progressive cone dystrophy (CORD5) maps to chromosome 17p12-p13. Genomics. 1995 Nov. 20; 30(2):281-6.; Becerra S P, et al., Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop. J Biol Chem. 1995 Oct. 27; 270(43):25992-9.; DiPaolo B R, et al., Identification of proteins differentially expressed in quiescent and proliferatively senescent fibroblast cultures. Exp Cell Res. 1995 September; 220(1):178-85.; Wu Y Q, et al., Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes. Protein Expr Purif. 1995 August; 6(4):447-56.; Tombran-Tink J, et al., Expression, secretion, and age-related down-regulation of pigment epithelium-derived factor, a serpin with neurotrophic activity. J Neurosci. 1995 July; 15(7 Pt 1):4992-5003.; Taniwaki T, et al., Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture. J Neurochem. 1995 June; 64(6):2509-17.; Pignolo R J, et al., Analysis of EPC-1 growth state-dependent expression, specificity, and conservation of related sequences. J Cell Physiol. 1995 January; 162(1):110-8.; Tombran-Tink J, et al., Localization of the gene for pigment epithelium-derived factor (PEDF) to chromosome 17p13.1 and expression in cultured human retinoblastoma cells. Genomics. 1994 Jan. 15; 19(2):266-72.; Seigel G M, et al., Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity. Growth Factors. 1994; 10(4):289-97.; Becerra S P, et al., Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. J Biol Chem. 1993 Nov. 5; 268(31):23148-56.; Pignolo R J, et al., Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state. J Biol Chem. 1993 Apr. 25; 268(12):8949-57.; Steele F R, et al., Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci USA. 1993 Feb. 15; 90(4)1526-30.; Tombran-Tink J, et al., PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity. Exp Eye Res. 1991 September; 53(3):411-4.; Seigel et al., Growth Factors, vol. 10, pp. 289-297, 1994.; Becerra et al., “Recombinant Human Fetal Retinal Pigment Epithelium-Derived Factor (PEDF),” Abstract 658-50, presented at Investigative Ophthalmology & Visual Science Annual Meeting (May 3-May 8, 1992).; Becerra et al., “A Novel Retinal Neurotrophic Factor (PEDF): A Serine Protease Inhibitor?” presented at NIH Research Festival 1992 (Sep. 21-25, 1992).; Becerra, et al., “Structure-Function Studies of Pigment Epithelium Derived Factor (PEDF),” The FASEB Journal (Abstract No. 192), vol. 7, No. 7, Apr. 20, 1993.; Pignolo, R. J., et al., “Senescent WI-38 Cells Fail To Express EPC-1, A Gene Induced In Young Cells Upon Entry Into The G.sub.0 State,” The Journal of Biological Chemistry, vol. 268, No. 12, Apr. 25, 1993, pp. 8949-8957.; Tombran-Tink et al., “RPE-54—A Unique RPE Product with Neuronal Differentiating Activity,” Investigative Ophthalmology & Visual Science, 29, 414 (1989).; Tombran-Tink et al., “Molecular Cloning and Chromoscomal Localization of the Gene for Human Pigment Epithelium-Derived Factor (PEDF),” Investigative Ophthalmology & Visual Science, 33(4), 828 (1992).; Tombran-Tink, et al., “Neurotrophic Activity of Interphotoreceptor Matrix on Human Y79 Retinoblastoma Cells,” The Journal of Comparative Neurology, 1992.; Zhigiang Zou, et al., “Maspin, A Serpin With Tumor-Suppressing Activity In Human Mammary Epithelial Cells,” Science, vol. 263, pp. 526-530, Jan. 28, 1994.

Metabolism of Glutathione.

The synthesis of GSH is dependent upon the availability of cysteine either supplied directly from the diet or cysteine or indirectly from methionine via the transsulfuration pathway. GSH synthesis and metabolism is governed by the enzymes of the γ-glutamyl cycle. GSH is synthesized intracellularly by the consecutive actions of γ-glutamylcysteinyl synthetase (Reaction 1) and GSH synthetase (Reaction 2). The action of the latter enzyme is feedback inhibited by GSH. The breakdown of GSH (and also of its oxidized form, GSSG) is catalyzed by γ-glutamyl transpeptidase, which catalyzes the transfer of the gamma-glutamyl moiety to acceptors such as sulfhydryl-containing amino acids, certain dipeptides, and GSH itself (Reaction 3). The cellular turnover of GSH is associated with its transport, in the form of GSH, across cell membranes, where the majority of the transpeptidase is found. During this transport, GSH interacts with γ-glutamyl transferase (also known as transpeptidase) to form γ-glutamyl amino acids which are transported into cells. Intracellular γ-glutamyl amino acids are substrates of γ-glutamyl cyclotransferase (Reaction 4) which converts these compounds into the corresponding amino acids and 5-oxo-L-proline. The ATP-dependent conversion of 5-L-oxoproline to L-glutamate is catalyzed by the intracellular enzyme 5-oxo-prolinase (Reaction 5). The cysteinylglycine formed in the transpeptidase reaction is split by dipeptidase (Reaction 6). These six reactions constitute the γ-glutamyl cycle, which accounts for the synthesis and enzymatic degradation of GSH.

Two of the enzymes of the cycle also function in the metabolism of S-substituted GSH derivatives, which may be formed nonenzymatically by reaction of GSH with certain electrophilic compounds or by GSH S-transferases (Reaction 7). The γ-glutamyl moiety of such conjugates is removed by the action of γ-glutamyl transpeptidase (Reaction 3), a reaction facilitated by γ-glutamyl amino acid formation. The resulting S-substituted cysteinylglycines are cleaved by dipeptidase (Reaction 6A) to yield the corresponding S-substituted cysteines, which may undergo N-acetylation (Reaction 8) or an additional transpeptidation reaction to form the corresponding γ-glutamyl derivative (Reaction 3A).

Intracellular GSH is converted to its oxidized, dimeric form (GSSG) by selenium-containing GSH peroxidase, which catalyzes the reduction of H₂O₂ and other peroxides (Reaction 9). GSH is also converted to GSSG by transhydrogenation (Reaction 10). Reduction of GSSG to GSH is mediated by the widely-distributed enzyme GSSG reductase which uses NADPH (Reaction 11). Extracellular conversion of GSH to GSSG has also been reported; the overall reaction requires O₂ and leads to the formation of H₂O₂ (Reaction 12). GSSG is also formed by reaction of GSH with free radicals. The glutathione-dependent antioxidant system consists of glutathione plus two enzymes: glutathione peroxidase and glutathione reductase. As this system operates, glutathione cycles between its oxidized (GSSG) and reduced (GSH) forms.

Lipid hydroperoxides, which are formed during the peroxidation of lipids containing unsaturated fatty acids, are reduced, not by the usual glutathione peroxidase, but by a special enzyme designed specifically to handle peroxidized fatty acids in phospholipids. This enzyme, known as phospholipid hydroperoxide glutathione peroxidase is protein that can reduce both H₂O₂ and lipid hydroperoxides to the corresponding hydroxides (water and a lipid hydroxide, respectively). In contrast to the phospholipid hydroperoxide glutathione peroxidase, ordinary glutathione peroxidase is unable to act on lipid hydroperoxides.

Transport of Glutathione.

The intracellular level of GSH in mammalian cells is in the range of 0.5-10 millimolar, while micromolar concentrations are typically found in blood plasma. Intracellular glutathione is normally over 99% reduced form (GSH). The normal healthy adult human liver synthesizes 8-10 grams of GSH daily. Normally, there is an appreciable flow of GSH from liver into plasma. The major organs involved in the inter-organ transport of GSH are the liver and the kidney, which is the primary organ for clearance of circulating GSH. It has been estimated to account for 50-67% of net plasma GSH turnover. Several investigators have found that during a single pass through the kidney, 80% or more of the plasma GSH is extracted, greatly exceeding the amount which could be accounted for by glomerular filtration. While the filtered GSH is degraded stepwise by the action of the brush-border enzymes γ-glutamyltransferase and cysteinylglycine dipeptidase, the remainder of the GSH appears to be transported via an unrelated, Na+-dependent system present in basal-lateral membranes.

GSH transported from hepatocytes interacts with the transpeptidase of ductile cells, and there appears to be a substantial reabsorption of metabolites by ductule endothelium. In the rat, about 12 and 4 nmoles/g/min of GSH appear in the hepatic vein and bile, respectively.

Glutathione exists in plasma in four forms: reduced glutathione (GSH), oxidized glutathione (GSSG), mixed disulfide with cysteine (CySSG) and protein bound through a sulfhydryl linkage (GSSPr). The distribution of glutathione equivalents is significantly different than that of cyst(e)ine, and when either GSH or cysteine is added at physiological concentration, a rapid redistribution occurs. The distribution of glutathione equivalents in rat plasma is 70.0% protein bound, with the remaining 30% apportioned as follows: 28.0% GSH, 9.5% GSSG, and 62.6% as the mixed disulfide with cysteine. The distribution of cysteine equivalents was found to be 23% protein bound, with the remaining 77% distributed as follows: 5.9% cysteine, 83.1% cystine, and 10.8% as the mixed disulfide with glutathione. Plasma thiols and disulfides are not in equilibrium, but appear to be in a steady state maintained in part by transport of these compounds between tissues during the interorgan phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in the analysis of transient changes in glutathione and cysteine. This buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins. In erythrocytes, GSH has been implicated in reactions which maintain the native structure of hemoglobin and of enzymes and membrane proteins. GSH is present in erythrocytes at levels 1000 times greater than in plasma. It functions as the major small molecule antioxidant defense against toxic free radicals, an inevitable by-product of the erythrocytes' handling of O₂.

Glutathione and the Immune System.

The importance of thiols and especially of GSH to lymphocyte function has been known for many years. Adequate concentrations of GSH are required for mixed lymphocyte reactions, T-cell proliferation, T- and B-cell differentiation, cytotoxic T-cell activity, and natural killer cell activity. Adequate GSH levels have been shown to be necessary for microtubule polymerization in neutrophils. Intraperitoneally administered GSH augments the activation of cytotoxic T-lymphocytes in mice, and dietary GSH was found to improve the splenic status of GSH in aging mice, and to enhance T-cell-mediated immune responses.

The presence of macrophages can cause a substantial increase of the intracellular GSH levels of activated lymphocytes in their vicinity. Macrophages consume cystine via a strong membrane transport system, and generate large amounts of cysteine which they release into the extracellular space. It has been demonstrated that macrophage GSH levels (and therefore cysteine equivalents) can be augmented by exogenous GSH. T-cells cannot produce their own cysteine, and it is required by T-cells as the rate-limiting precursor of GSH synthesis. The intracellular GSH level and the DNA synthesis activity in mitogenically-stimulated lymphocytes are strongly increased by exogenous cysteine, but not cystine. In T-cells, the membrane transport activity for cystine is ten-fold lower than that for cysteine. As a consequence, T-cells have a low baseline supply of cysteine, even under healthy physiological conditions. The cysteine supply function of the macrophages is an important part of the mechanism which enables T-cells to shift from a GSH-poor to a GSH-rich state.

The importance of the intracellular GSH concentration for the activation of T-cells is well established. It has been reported that GSH levels in T-cells rise after treatment with GSH; it is unclear whether this increase is due to uptake of the intact GSH or via extracellular breakdown, transport of breakdown products, and subsequent intracellular GSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cell activation in vitro. Depletion of intracellular GSH has been shown to inhibit the mitogenically-induced nuclear size transformation in the early phase of the response. Cysteine and GSH depletion also affects the function of activated T-cells, such as cycling T-cell clones and activated cytotoxic T-lymphocyte precursor cells in the late phase of the allogenic mixed lymphocyte culture. DNA synthesis and protein synthesis in IL-2 dependent T-cell clones, as well as the continued growth of preactivated CTL precursor cells and/or their functional differentiation into cytotoxic effector cells are strongly sensitive to GSH depletion.

The activation of physiologic activity of mouse cytotoxic T-lymphocytes in vivo was found to be augmented by intraperitoneal (i.p.) GSH in the late phase but not in the early phase of the response. The injection of GSH on the third day post immunization mediated a 5-fold augmentation of cytotoxic activity. Dietary GSH supplementation can reverse age-associated decline of immune response in rats, as demonstrated by maintenance of Concanavalin A stimulated proliferation of splenocytes in older rats.

Glutathione status is a major determinant of protection against oxidative injury. GSH acts on the one hand by reducing hydrogen peroxide and organic hydroperoxides in reactions catalyzed by glutathione peroxidases, and on the other hand by conjugating with electrophilic xenobiotic intermediates capable of inducing oxidant stress. The epithelial cells of the renal tubule have a high concentration of GSH, no doubt because the kidneys function in toxin and waste elimination, and the epithelium of the renal tubule is exposed to a variety of toxic compounds. GSH, transported into cells from the extracellular medium, substantially protects isolated cells from intestine and lung are against t-butylhydroperoxide, menadione or paraquat-induced toxicity. Isolated kidney cells also transport GSH, which can supplement endogenous synthesis of GSH to protect against oxidant injury. Hepatic GSH content has also been reported to rise, indeed to double, in the presence of exogenous GSH. This may be due either to direct transport, as has been reported for intestinal and alveolar cells, or via extracellular degradation, transport, and intracellular resynthesis.

The nucleophilic sulfur atom of the cysteine moiety of GSH serves as a mechanism to protect cells from harmful effects induced by toxic electrophiles. The concept that glutathione S-conjugate biosynthesis is an important mechanism of drug and chemical detoxification is well established. GSH conjugation of a substrate generally requires both GSH and glutathione-S-transferase activity. The existence of multiple glutathione-S-transferases with specific, but also overlapping, substrate specificities enables the enzyme system to handle a wide range of compounds.

Several classes of compounds are believed to be converted by glutathione conjugate formation to toxic metabolites. Halogenated alkenes, hydroquinones, and quinones have been shown to form toxic metabolites via the formation of S-conjugates with GSH. The kidney is the main target organ for compounds metabolized by this pathway. Selective toxicity to the kidney is the result of the kidney's ability to accumulate intermediates formed by the processing of S-conjugates in the proximal tubular cells, and to bioactivate these intermediates to toxic metabolites.

The administration of morphine and related compounds to rats and mice results in a loss of up to approximately 50% of hepatic GSH. Morphine is known to be biotransformed into morphinone, a highly hepatotoxic compound, which is 9 times more toxic than morphine in mouse by subcutaneous injection, by morphine 6-dehydrogenase activity. Morphinone possesses an α,β-unsaturated ketone, which allows it to form a glutathione S-conjugate. The formation of this conjugate correlates with loss of cellular GSH. This pathway represents the main detoxification process for morphine. Pretreatment with GSH protects against morphine-induced lethality in the mouse.

The deleterious effects of methylmercury on mouse neuroblastoma cells are largely prevented by coadministration of GSH. GSH may complex with methylmercury, prevent its transport into the cell, and increase cellular antioxidant capabilities to prevent cell damage. Methylmercury is believed to exert its deleterious effects on cellular microtubules via oxidation of tubulin sulfhydryls, and by alterations due to peroxidative injury. GSH also protects against poisoning by other heavy metals such as nickel and cadmium.

Because of its known role in renal detoxification and its low toxicity, GSH has been explored as an adjunct therapy for patients undergoing cancer chemotherapy with nephrotoxic agents such as cisplatin, in order to reduce systemic toxicity. In one study, GSH was administered intravenously to patients with advanced neoplastic disease, in two divided doses of 2,500 mg, shortly before and after doses of cyclophosphamide. GSH was well-tolerated and did not produce unexpected toxicity. The lack of bladder damage, including microscopic hematuria, supports the protective role of this compound. Other studies have shown that i.v. GSH coadministration with cisplatin and/or cyclophosphamide combination therapy, reduces associated nephrotoxicity, while not unduly interfering with the desired cytotoxic effect of these drugs. See: Bohm, S., Battista-Spatti, G., DiRe, F., Oriana, S., Pilotti, S., Tedeschi, M., Tognella, S. & Zunino, F.: A feasibility study of cisplatin administration with low-volume hydration and glutathione protection in the treatment of ovarian carcinoma. Anticancer Res. 11:1613-1616, 1991.; Cozzaglio, L., Doci, R., Colla, G., Zunino, F., Casciarri, G. & Gennari, L.: A feasibility study of high-dose cisplatin and 5-fluorouracil with glutathione protection in the treatment of advanced colorectal cancer. Tumori 76:590-594, 1990.; Di Re, F., Bohm, S., Oriana, S., Spatti, G. B., & Zunino, F.: Efficacy and safety of high-dose cisplatin and cyclophosphamide with glutathione protection in the treatment of bulky advanced epithelial ovarian cancer. Cancer Chemother. Pharmacol. 25:355-360, 1990.; Nobile, M. T., Vidili, M. G., Benasso, M., Venturini, M., Tedeschi, M., Zunino, F., & Rosso, R.: A preliminary clinical study of cyclophosphamide with reduced glutathione as uroprotector. Tumori 75:257-258, 1989.

Clinical Use of Glutathione

Ten elderly patients with normal glucose tolerance and ten elderly patients with impaired glucose tolerance (IGT) underwent GSH infusion, 10 mg/min for 120 min, for a total dose of 1,200 mg in 2 hr, under basal conditions and during 75 g oral glucose tolerance tests and intravenous glucose tolerance tests. Basal plasma total glutathione levels were essentially the same for normal and IGT groups, and GSH infusion under basal conditions increased GSH to similar levels. This study demonstrated that GSH significantly potentiated glucose-induced insulin secretion in patients with IGT. No effect was seen on insulin clearance and action.

The antihypertensive effect of an i.v. bolus of 1,844 mg. or 3,688 mg. GSH was studied in normal and mild to moderate essential hypertensive subjects and in both hypertensive and non-hypertensive diabetics, both type I and type II. The administration of 1,844 mg. GSH produced a rapid and significant decrease in both systolic and diastolic blood pressure, within ten minutes, but which returned to baseline within 30 minutes, in both groups of hypertensive patients and in non-hypertensive diabetics, but had no effect in normal healthy subjects. At the 3,699 mg. dose, not only did the blood pressure decrease in the hypertensive subjects, but GSH produced a significant decrease in the blood pressure values in normal subjects as well.

GSH, 1,200 mg/day intravenously administered to chronic renal failure patients on hemodialysis was found to significantly increase studied hematologic parameters (hematocrit, hemoglobin, blood count) as compared to baseline, and holds promise to reverse the anemia seen in these patients. See, Costagliola, C., Romano, L., Scibelli, G., de Vincentiis, A., Sorice, P. & DiBenidetto, A.: Anemia and chronic renal failure: a therapeutic approach by reduced glutathione parenteral administration. Nephron 61:404-408, 1992.

Toxicological Effects of Glutathione.

The reported LD₅₀ of GSH in rats and mice via various routes of administration are listed in the table below. GSH has an extremely low toxicity, and oral LD₅₀ measurements are difficult to perform due to the sheer mass of GSH which has to be ingested by the animal in order to see any toxic effects.

Route of Animal Admin. LD₅₀ Reference Mouse Oral 5000 mg/kg Modern Pharmaceuticals of Japan, IV Edition, Tokyo, Japan Pharmaceutical, Medical and Dental Supply Exporters′ Association, 1972, p 93. Mouse Intraperitoneal 4020 mg/kg Modern Pharmaceuticals of Japan, IV Edition, Tokyo, Japan Pharmaceutical, Medical and Dental Supply Exporters′ Association, 1972, p 93. Mouse Intraperitoneal 6815 mg/kg Toxicology, vol. 62, p. 205, 1990. Mouse Subcutaneous 5000 mg/kg Modern Pharmaceuticals of Japan, IV Edition, Tokyo, Japan Pharmaceutical, Medical and Dental Supply Exporters′ Association, 1972, p 93. Mouse Intravenous 2238 mg/kg Japanese J. of Antibiotics, vol. 38, p. 137, 1985. Mouse Intramuscular 4000 mg/kg Modern Pharmaceuticals of Japan, III Edition, Tokyo, Japan Pharmaceutical, Medical and Dental Supply Exporters′ Association, 1968, p 97.

GSH can be toxic, especially in cases of ascorbate deficiency, and these effects may be demonstrated in, for example, ascorbate deficient guinea pigs given 3.75 mmol/kg daily (1,152 mg/kg daily) in three divided doses, whereas in non-ascorbate deficient animals, toxicity was not seen at this dose, but were seen at double this dose. See: Dalhoff, K., Ranek, L., Mantoni, M. & Poulsen, H. E.: Glutathione treatment of hepatocellular carcinoma. Liver 12:341-343, 1992.; Dekant, W.: Bioactivation of nephrotoxins and renal carcinogens by glutathione S-conjugate formation. Toxicol. Letters 67:151-60, 1993.; Domingo, J. L., Gomez, M., Llobet, J. M. & Corbella, J.: Chelating agents in the treatment of acute vanadyl sulphate intoxication in mice. Toxicology 62: 203-211, 1990.; Martensson, J., Han, J., Griffith, O. W. & Meister, A.: Glutathione ester delays the onset of scurvy in ascorbate-deficient guinea pigs. Proc. Nat. Acad. Sci. USA 90:317-321, 1993.; Thust, R, and Bach, B.: Exogenous glutathione induces sister chromatid exchanges, clastogenicity and endoreduplication in V79-E Chinese hamster cells. Cell Biol. Toxicol. 1:123-31, 1985.; Aebi, S. & Lauterberg, B. H.: Divergent effects of intravenous GSH and cysteine on renal and hepatic GSH. Aer. J. Physiol. 263(2 pt 2):R348-R352, 1992.; Ammon, H. P. T., Melien, M. C. M. & Verspohl, E. J.: Pharmacokinetics of intravenously administered glutathione in the rat. J. Pharm. Pharmacol. 38:721-725, 1986.; Anderson, M. E., Powrie, F., Puri, R. N., & Meister, A.: Glutathione monoethyl ester: Preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239:538-548, 1985.; Aw, T. Y., Wierzbicka, G. & Jones, D. P.: Oral glutathione increases tissue glutathione in vivo. Chem. Biol. Interact. 80:89-97, 1991.; Borok, Z., Buhl, R., Grimes, G J., Bokser, A. D., Hubbard, R. C., Holroyd, K J., Roum, J. H., Czerski, D. B., Cantin, A. M., & Crystal, R. G.: Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. The Lancet 338:215-216, 1991.; Buhl, R., Vogelmeier, C., Critenden, M., Hubbard, R. C., Hoyt, Jr., R. F., Wilson, E. M., Cantin, A. M. & Crystal, R. G.: Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc. Natl. Acad. Sci. USA 87: 4063-4067, 1990.; Bump, E. A., al-Sarraf, R., Pierce, S. M. & Coleman, C. N.: Elevation of mouse kidney thiol content following administration of glutathione. Radiother. Oncol. 23:21-25, 1992.; Griffith, O. W., Bridges, R. J., & Meister, A.: Formation of g-glutamyl-cyst(e)ine in vivo is catalyzed by γ-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 78:2777-2781, 1981.; Hagen, T. M., Wierzbicka, G. T., Bowman, B. B., Aw, T. Y. & Jones, D. P.: Fate of dietary glutathione. Disposition in the gastrointestinal tract. Am. J. Physiol. 259: G530-G535, 1990.; Hagen, T. M. & Jones, D. P.: Transepithelial transport of glutathione in vascularly perfused small intestine of rat. Am. J. Physiol. 252:G607-G613, 1987.; Hagen, T. M., Wierzbicka, G. T., Sillau, A. H., Bowman, B. B. & Jones, D. P.: Bioavailability of dietary glutathione. Effect on plasma concentration. Am. J. Physiol. 259:G524-G529, 1990.; Hahn, R., Wendel, A. & Flohé, L.: The fate of extracellular glutathione in the rat. Biochim. Biophys. Acta 539:324-337, 1978.; Puri, R. N., & Meister, A.: Transport of glutathione, as g-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Natl. Acad. Sci. USA 80:5258-5260, 1983.; Vina, J., Perez, C., Furukawa, T., Palacin, M. & Vina, J. R.: Effect of oral glutathione on hepatic glutathione levels in rats and mice. Brit. J. Nutr. 62:683-91, 1989.; Aebi, S., Asserto, R., & Lauterberg, B. H.: High-dose intravenous glutathione in man.: Pharmacokinetics and effects on cyst(e)ine levels in plasma and urine. Eur. J. Clin. Invest. 21:103-110, 1991.; Hagen, T. M. and Jones, D. P. Role of glutathione transport in extrahepatic detoxication. in Glutathione Centennial: Molecular Perspectives and Clinical Implications, N. Taniguchi, T. Higashi, Y. Sakamoto and A. Meister, eds. Acad. Press, New York, 1990.; Jones, D. P., Hagen, T. M., Weber, R., Wierzbicka, G. T., and Bonkovsky, H. L.: Oral administration of glutathione (GSH) increases plasma GSH concentration in humans. FASEB J. 3:A1250 (5953), 1990.

Effects of Glutathione on the Circulatory System

Glutathione impacts many aspects of the circulatory system, including interactions with nitric oxide signaling, ischemia, and control over vascular endothelium. See: Demopoulos, H. B., Flamm, E. S., Pietronigro, D. D., and Seligman, M. L.: Free radical pathology and antioxidants in regional cerebral ischemia and central nervous system trauma. In: Anesthesia and Neurosurgery, eds. J. E. Cottrell and H. Tunndorf. C. V. Mosby, St. Louis, 1986, pp. 246-279.; Kagan, V. E., Bakalova, R. A., Koynova, G. M., Tyurin, V. A., Seriniva, E. A., Petkov, V. V., Staneva, D. S. and Packer, L.: Antioxidant protection of the brain against oxidative stress. In: Free Radicals in the Brain, eds. L. Packer, L. Prilipko, and Y. Christen. Springer-Verlag, New York, 1992, pp. 49-61.; Pietronigro, D. D., Demopoulos, H. B., Hovsepian, M. and Flamm, E. S.: Brain ascorbic acid depletion during cerebral ischemia. Stroke 13:117-119, 1982.; Shan, X., Aw, T. Y. and Jones, D. P.: Glutathione-dependent protection against oxidative injury. Pharmac. Ther. 47:61-71, 1990.; Simon, D. I., Stamler, J. S., Jaraki, O., et al.: Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor. Arterioscler. Thromb. 13(6):791-799, 1993.; Taccone-Gallucci, M., Lubrano, R., Clerico, A., Meloni, C., Morosetti, M., Meschini, L., Elli, M., Trapasso, E., Castello, M. A. & Casciani, C. U.: Administration of GSH has no influence on the RBC membrane: Oxidative damage to patients on hemodialysis. ASAIO Journal 38:855-857, 1992.

Use of High-Dose Oral GSH in Cancer Patients.

In one published study, eight patients with hepatocellular carcinoma were treated with 5 g oral reduced glutathione per day. Two patients withdrew shortly after receiving GSH due to intolerable side-effects (gastrointestinal irritation and sulfur odor). The remaining patients, aged 27-63, three male and three female, did not experience side-effects from this high dose of GSH and continued to take 5 g oral GSH for periods ranging from 119 days (at which time the patient died from her tumor) to >820 days (this patient was still alive at the time of publication and was still taking 5 g oral GSH daily; his tumor had not progressed and his general condition was good). Two of the female patients survived 1 year and exhibited regression or stagnation of their tumor growth. The remaining two patients, both male, died as expected within 6 months.

Experience in HIV-Infected Patients.

A commercially available nutritional formulation containing 3 grams of reduced glutathione was given daily to a group of 46 AIDS patients for a period of three months by a group of private physicians. No significant GSH-related adverse effects were reported. No evidence of toxicities from laboratory studies or from clinical examinations was reported; however, no benefit was conclusively demonstrated.

Pharmacokinetics of Glutathione

The pharmacokinetics of intravenously administered GSH were determined in the rat and interpreted by means of an open, two-compartment model. Following a bolus injection of 50-300 mmol/kg GSH, arterial plasma concentrations of (i) GSH, (ii) oxidized glutathione/GSSG, (iii) total thiols, and (iv) soluble thiols minus GSH, were elevated and then rapidly decreased non-exponentially, as anticipated. With increasing dose, the rate constant for drug elimination and plasma clearance increased form 0.84 to 2.44 mL/min. and the half-life of the elimination phase decreased from 52.4 to 11.4 minutes. Both the apparent volume of distribution and the degree of penetration of GSH into the tissues were diminished with increasing dose (from 3.78 to 1.33 L/Kg and from 6.0 to 0.51 as k₁₂/k₂₁, respectively). The data indicate that GSH is rapidly eliminated. This is mainly due to rapid oxidation in plasma rather than by increased tissue extraction or volume distribution. Thus, plasma GSH levels appear to be quickly regulated by which the body may maintain concentrations within narrow physiological limits.

When single doses of 600 mg GSH were administered intravenously to sheep, GSH levels in venous plasma and lung lymph rose transiently. The mean concentration was approximately 50 mM for venous plasma, peaking at 30 min, and returning to baseline after 45 minutes. Lung lymph peak level was about 100 mM at 15 min, returning to baseline after 30 minutes. Average epithelial lining fluid (ELF) levels were variable but showed no significant increase over baseline during the three hour observation period. Urine excretion was rapid with peak levels at 15 minutes. In both plasma and lung lymph, GSH accounted for greater than 95% of the total glutathione (GSH plus GSSG). In ELF 75.4% of the baseline glutathione was in the reduced form, whereas in urine 59.6% was present as GSH.

Orally ingested reduced glutathione is absorbed intact from the small intestine in a rat model, specifically in the upper jejunum. It is noted that rat metabolism differs from man, and therefore the results of rat studies should be verified in man before the results are extrapolated. Plasma GSH concentration in rats increased from 15 to 30 mM after administration of GSH either as a liquid bolus (30 mM) or mixed (2.5-50 mg/g) in AIN-70 semi-synthetic diet (11). GSH concentration was maximal at 90-120 minutes after GSH administration and remained high for over 3 hours. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and re-synthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine (BSO) and acivicin showed that the increased plasma GSH came mostly from absorption of intact GSH instead of from its metabolism. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus, dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH.

Administration of oral GSH increased hepatic glutathione levels in: (i) rats fasted 48 hours, (ii) mice treated with GSH depletors, and (iii) mice treated with paracetamol (a drug which promotes a depletion of hepatic GSH followed by hepatic centrilobular necrosis). In these experiments, the animals were orally intubated with 1000 mg/kg body weight GSH. Mean pretreatment values in 48-hour fasted rats were 3.0-3.1 mmol/g fresh hepatic tissue. Mean values after treatment were 5.8, 4.2, and 7.0 mmol/g fresh hepatic tissue for 2.5, 10, and 24 hours post-treatment, respectively. Mice were given an oral dose of GSH (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 hr in liver, kidney, heart, lung, brain, small intestine and skin. GSH concentrations in plasma increased from 30 mM to 75 mM within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. No increases over control values were obtained in most tissues except lung over the same time course. Mice pretreated with the GSH synthesis inhibitor BSO had substantially decreased tissue concentrations of GSH, and oral administration of GSH to these animals resulted in statistically-significant increases in the GSH concentrations of kidney, heart, lung, brain, small intestine and skin but not in liver. See: Fahey, R. C., and Newton, G. L.: Determination of low molecular weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Meth. Enzymol. 143:85-96, 1987. See: Mills, B. J., Richie. J. P. Jr., and Lang, C. A.: Sample processing alters glutathione and cysteine values in blood. Anal. Biochem. 184:263-267, 1990.; Richie, J. P. Jr., and Lang. C. A.: The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection. Anal. Biochem. 163:9-15, 1987.; Tietz, F.: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27:502-22, 1969.

The kinetics and the effect of glutathione on plasma and urine sulfhydryls were studied in ten healthy human volunteers. Following the intravenous infusion of 2000 mg/m² of GSH the concentration of total glutathione in plasma increased from 17.5-13.4 mmol/Liter (mean=/−SD) to 823-326 mmol/Liter. The volume of distribution of exogenous glutathione was 176-107 MI/Kg and the elimination rate constant was 0.063-0.027/minute, corresponding to a half-life of 14.1-9.2 minutes. Cysteine in plasma increased from 8.9-3.5 mmol/Liter to 114-45 mmol/Liter after the infusion. In spite of the increase in cysteine, the plasma concentration of total cyst(e)ine (i.e. cysteine, cystine, and mixed disulfides) decreased, suggesting an increased uptake of cysteine from plasma into cells. The urinary excretion of glutathione and of cyst(e)ine was increased 300-fold and 10-fold respectively, in the 90 minutes following the infusion.

Normal healthy volunteers were given an oral dose of GSH to determine whether dietary GSH could raise plasma GSH levels. Results showed that an oral dose of GSH (15 mg/kg) raised plasma glutathione levels in humans 1.5-10 fold over the basal concentration in four out of five subjects tested, with a mean value three times that of normal plasma GSH levels. Plasma GSH became maximal 1 hour after oral administration, dropping to approximately ½ maximal values after three hours. Equivalent amounts of GSH amino acid constituents failed to increase plasma levels of GSH. GSH bound to plasma proteins also increased with the same time course as seen with free GSH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As apparent from the discussion above, there are a wide variety of pathologic and degenerative disorders which are impacted by either the level of glutathione and/or ratio of oxidized to reduce species, or secondary influences based on said level or ratio. In general, it is believed that, within a pharmaceutically acceptable range, as determined by toxic ranges, increased levels of glutathione tend to improve health, and low oxidized to reduced ratios are beneficial. Thus, the present invention advocates administration of reduced glutathione for both healthy persons, which are all subject to aging and constant oxidative insult, and persons suffering from pathology as discussed above. As shown, glutathione can itself be administered orally with good bioavailability, and therefore this mode of administration is preferred. It is, of course possible, to administer pharmaceutically acceptable derivatives of glutathione or use other routes of administration. Likewise, Glutathione may be administered alone or in combination with an additive or synergistic composition, or to remediate toxicity of a co-administered agent.

There is supportive literature for the use of Glutathione, and other sulfhydryl-antioxidants) demonstrating: (i) immediate radiation protection, safely; (ii) rapid inactivation of sulfur mustard gas, safely; (iii) immediate modification of the three exotoxins of anthrax, safely, such that the formation of either of the two heterotoxins becomes impossible and the toxins are muted, thereby preventing cell entry and the subsequent cytokines; (iv) rapid protection vs. Sepsis, as occurs with serious complications following smallpox vaccination, smallpox infection, burns and trauma, among others.

Thus, Glutathione is useful against a variety of nuclear, biological and chemical agents, and indeed its broad spectrum activity makes it particularly useful as a “first line” defense against unknown ill-defined threats. Its low toxicity also permits long term prophylactic use, during times of alert or imminent threat.

Glutathione is available in a formulation which renders this otherwise difficult molecule safe, stable, and bioavailable (80% is absorbed and intracellular in 1.5 hours following oral dosing) with dose responsive pharmacokinetics. In Phase 1 studies on non symptomatic HIV positive people, the inherent safety of this naturally produced substance (10 grams/24 hours) was affirmed for the Anti Viral Division, of the FDA.

Glutathione is useful to combat and protect against unusual microbal agents, sepsis, anthrax toxins, small pox vaccination complications, smallpox, and genetically engineered smallpox carrying copies of the Interleukin 4 (IL-4) gene, (based on glutathione (GSH) suppression of the oxidative stress; GSH suppression of excess cytokine production; GSH suppression of the excess prostaglandin synthesis; GSH suppression of viral replication at the level of the proviral DNA integrated in host cells; GSH up-regulation of T helper 1 response patterns and down regulation of T helper 2 response patterns which includes IL-4). Sepsis kills more people than all the cancers of the lung, breast, prostate and colon, combined, and also results in extraordinary costs in the Intensive Care Units; biowarfare microbes, like smallpox, kill as a result of the Sepsis induced. Glutathione is also useful in the treatment of radiologic and chemical injuries, and as prophylaxis against prospective exposure.

Glutathione is also useful to treat prevalent neurologic disorders that are pharmaceutically underserved: Alzheimer's and Parkinson's.

Glutathione (“GSH”) is useful for its properties of safely neutralizing poisonous chemicals, like sulfur mustard gas, and radioactive substances. The supporting literature also indicates that glutathione has the ability to stem the lethal “cytokine storms” of rampant inflammation seen with anthrax toxins, smallpox vaccination complications, smallpox infections, and infections with Interleukin 4 (IL-4) enhanced smallpox and other “exotic microbes”.

The preferred formulation and method of administering GSH, directly and rapidly replenishes the body's stores of bioprotectant, glutathione. Cellular concentrations of this substance are vulnerable to erosion by many factors including age, diabetes, smoking, infections and exposures to toxins and radioactive compositions. It is essential to ensure that glutathione levels are kept at high concentrations, in order to be protective, and to allow glutathione to carry out its regulatory processes in cells . . . it is the most crucial biochemical in a cell . . . life processes cease rapidly without it. Glutathione can be deployed easily, to help protect military personnel and civilians from wartime and terrorist dispersions of poison gas, radioactive materials, and lethal microbes.

Glutathione levels can be restored and augmented, inside cells, starting approximately 30 minutes after oral administration. This is well within the “window of therapy” for most of the survivable exposures. The large safety factor of oral glutathione, and the proven high acceptability of restorative/augmentive regimens (4 to 9 grams/day for 12 months) makes it possible to enhance and maintain high glutathione levels, ahead of time, among first responders, and special military personnel on the ground. These available regimens appear to be sufficient since normal, prime production is 10 grams per 24 hours, in humans. There are no significant toxicities.

The drug is preferably provided as the stabilized, pharmaceutical glutathione, in powder form, readily dissolvable in any convenient fluid. The taste is benign and citrus-like. Shelf life of the powder is 2.5 years. It is also available in a more costly, encapsulated dosage form.

The ease, safety and high acceptability of the regimen make it practical to treat radiation survivors at long term risk for cancer, on a continuous multi-year regimen. Cancer risks among such survivors is one of the major considerations in the aftermath of radiation exposures. Glutathione, aside from its immediate actions in neutralizing toxic free radicals caused by radiation, also is an essential cell regulator that helps control several classes of genes, including suppressing genes, such as the Ras family of oncogenes, that promote growth of cancer cells, and genes that promote growth of new blood vessels that cancers need for growth and spread. These long term properties of glutathione are thought to help in decreasing the risks of cancer, and may offer synergistic approaches to the cancer treatment.

Glutathione (GSH) is a tripeptide composed of three unusually linked amino acids, the middle one of which has a well positioned sulfur-hydrogen (—SH) group “sculpted” by eons of evolutionary biochemistry. It is a central cell regulator at the biochemical epicenter of most life processes.

GSH is a major bioprotectant substance in cells. It combines with many chemicals that do not belong, and carries them out as excretory products, for example, sulfur mustard, or internalized uranium and plutonium.

GSH is an integral part of the physiology of the Immune System; depletions of GSH impair this and permit fulminating infections.

GSH stems the molecular pathology of anthrax and of Sepsis as seen in certain viral infections characterized by intense inflammation, such as smallpox.

GSH turns on or off regulatory proteins and genes by setting the redox potential.

GSH, in concert with specific enzymes (peroxidases and transferases), dismantles toxic fat molecules that have become peroxidized (rancid). This class of toxic substances causes inflammation and immune suppression.

GSH destroys the hydrogen peroxide (H₂O₂) routinely formed in mitochondria, the cell “furnaces” that efficiently produce energy for cell functions. Without sufficient GSH the mitochondria become “executioners” and induce unscheduled apoptotic cell death in strategic cells by leaking cytochrome c and caspaces via sulfhydryl-sensitive pores (permeability transition pores). This property of maintaining mitochondria is central in treating Parkinson's and Alzheimer's diseases.

GSH inhibits the elaboration of excessive growth factors, and blood vessel growth factors found in cancers.

GSH stems the activation of transcription factors, such as the NFκB's, that switch on disease-linked genes, as in inflammatory disorders, Sepsis, infections and cancers. Suppressing NFκB helps to curb the inflammation from sulfur mustard gas, anthrax, Sepsis, and smallpox.

GSH is critical for phagocytic cells that are essential to clear away debris and microbes in tissues. High GSH levels in macrophages enhance local tissue defenses against microbes. This is a factor in the rationale for using GSH to combat microbes used in Biologic Warfare, and microbes causing Sepsis.

High GSH concentrations are needed during antigen processing by macrophages, dendritic cells and B type lymphocytes, to maintain a productive balance between the T helper 1 (Th1) and Th2, response patterns which are reciprocally related. GSH up-regulates Th1 and down regulates Th2 response patterns. This is relevant to decreasing the life threatening risks of smallpox immunization and of potential import in countering IL-4 enhanced viruses (e.g. IL-4 enhanced smallpox)

The GSH properties of setting the redox potential, dismantling lipid hydroperoxides (LOOH's), neutralizing reactive oxygen species (“ROS”) and reactive nitrogen species (“RNS”) help to keep the family of NFκB transcription factors inactive, and out of the nucleus. The NFκB family of transcription factors activate several groups of genomic sequences, including proviral DNA sequences that have been incorporated in the infected host cells (e.g., HIV proviral DNA has been successfully studied in a number of peer-reviewed publications; it is likely that other, incorporated viral DNA sequences, like those of smallpox, may share a similar dependence on NFκB translocation into the nucleus and subsequent activation-binding to the proviral DNA).

GSH controls arachidonic acid metabolism and the products of the cyclo-oxygenases and lipoxygenases. These are pro-inflammatory, stimulate DNA synthesis and are also immuno-suppressive. This why cyclo-oxygenase-2 inhibitors (e.g. Vioxx) are anti-inflammatory and also decrease cancer risks.

One aspect of the present invention provides a method for the treatment of smallpox infections. While smallpox is a terrifying scourge, having killed 500 million people in the 20th Century, the molecular pathologic mechanisms have been defined, and high dose glutathione regimens pose a block vs. these mechanisms. The major organ involved in smallpox is the skin, with massive numbers of ulcerated pox lesions that may become secondarily infected. It is partially analogous to a patient with infected, third degree burns covering 80%-90% of the body, except smallpox is worse in that its inflammation assures the destruction of dermal GSH and GSH in macrophages and white blood cells. The basic pathology is analogous to that of Burns and Sepsis, but with added large numbers of circulating antigens, antigen antibody complexes, and cytokines. Glutathione (“GSH”) levels in most cells are not measurable in severe Sepsis and Burns. The GSH losses are profound and well documented in the Burns and Sepsis literature. “Cytokine Storms” characterize some of the pathology, and it is feasible to down regulate inflammatory cytokine production by inactivating Nuclear Factor κB (“NFκB”) with GSH. This works in inflammatory situations characterized by excess activation of NFκB.

GSH may also hinder the replication of the smallpox virus because GSH fosters the T helper 1 (“Th1”) response patterns that include IL-12 and Interferon gamma (INF γ) production . . . one of the body's most potent antivirals. GSH is known to up-regulate Th1 responses, which then reciprocally hinder Th2 responses, thereby hindering IL-4 production, a Th2 cytokine that can blunt INF γ production, and foster unbridled viral replication.

Basically, high dose therapy of smallpox infection with Rx GSH may blunt the excess NFκB activation, which will decrease the production of the toxic, inflammatory cytokines. Also, Rx GSH can foster Th1 responses and up-regulate INF γ production, a potent antiviral. When Th1 patterns are up-regulated, the reciprocal down-regulation of IL-4 is helpful, since IL-4 would otherwise interfere with INF γ. Through NFκB, GSH is also likely to have anti viral properties that would hinder replication of the smallpox virus, similar to how Rx GSH significantly inhibits HIV replication from its proviral DNA.

Pox viruses have been genetically engineered with IL-4 genes in rodents, and possibly in smallpox strains as well. In animal models, IL-4 enhanced pox viruses have proven lethal, despite successful immunization vs. the particular, natural pox virus. Among the pathologic consequences of excess IL-4, by such “super pox” viruses, is the suppression of INF γ. Rx GSH may be able to overcome this molecular pathology.

Another aspect of the present invention provides a method designed to help protect against the chemical, sulfur mustard gas, an inexpensive, easily prepared, dangerous substance that has been used in wars, and is thought to be a “favorite” of some terrorist groups. Accordingly, oral administration of GSH according to the present invention can restore and raise GSH levels inside cells, to safely help protect against this chemical. The blistering by this poison gas is not immediate, and its presence becomes obvious because of its odor. There is, therefore, time to orally administer GSH. It starts to raise cellular levels in 30 minutes, well within the “window of therapy”.

A further aspect of the present invention provides a method for protection against radioactive dispersions. This has significant utility not only for Counterterrorism, but also in peacetime uses in the U.S. and in countries like Japan and France with their heavy reliance on nuclear power for energy generation. Accidents, contamination, and waste problems continuously expose people, in the U.S. and globally. In the case of significant radiation exposures, glutathione can safely be used as a multi-year therapy to lower the risk of cancers, a major concern in radiation survivors.

A still further aspect of the present invention provides a method to help control anthrax infections and the dangerous, lingering effects of three toxins. These are three proteins and specific combinations of these must form chemically in order for the toxins to penetrate into cells wherein they cause the cell to produce prodigious amounts of injurious cytokines. Glutathione has the ability to prevent the formation of penetrating toxins, and it has the ability to slow down cytokine production through its gene suppressing functions. Patients can die even with appropriate use of doxycycline and ciprofloxacin, because of the exotoxins. GSH may help in this regard.

Because GSH is proven safe, and is a natural protectant produced by the body, it can be taken orally routinely, on a daily basis, to provide consistently high levels in all cells throughout the body; therefore, first responders and people in high risk situations can be continuously maintained at high glutathione levels. There are no toxicities, and the majority of individuals using high doses report positive salutary effects. The health benefits of GSH are broad. Basically, there is no “downside” to having people become well endowed with glutathione.

It is well known from recent literature that GSH synthesis falls after 45 years of age. At all ages it can be destroyed by excess of alcohol, dietary fat, carbohydrates, tobacco, sun, and aerobics.

Alzheimer's Disease is believed to represent a gradual loss of brain cells by the process of apoptosis, a form of cell death wherein the cell breaks up into fragments. This process is believed to start with a depletion of glutathione within the mitochondria, the “power plants” of cells. Some brain cells have several hundred mitochondria per cell because of their high energy requirements. If mitochondria are deficient in glutathione, energy production fades, and in addition, the mitochondria release highly destructive biochemicals that initiate apoptosis. Medical scientists in this field believe effective, safe restoration of glutathione in the brain could blunt the progression of Alzheimer's disease, particularly if detected and treated in the early stages.

Microvascular pathology has also been delineated in the Dementias, and contributes to perpetual, pathologic cycles. The microcirculation in the cerebrum of animal models was first defined with Scanning Electron Microscopy by H. B. Demopoulos and colleagues. Rx GSH counters this type of microvascular pathology by maintaining PGI2 synthesis in endothelium, and ensuring the desirable physiologic properties of vascular nitric oxide (.NO).

New methodologies that combine psychometrics (the ability of the patient to recite, verbatim, as much of a paragraph read to him/her, and other standardized short term memory tests) and brain imaging can detect early Alzheimer's and also chart the progression of this disorder.

These advances in modern Neurology make it feasible to conduct a clinical trial with early Alzheimer's patients, with placebo and treated groups, double blind, and randomized. It would be possible to follow the two groups and to compare the rates of progression to determine statistically significant differences. It is believed that Glutathione can intercede and slow or stop this process, and even allow a return of some function, in patients suffering from chronic dementias associated with progressive apoptosis of brain cells. Other chronic diseases associated with progressive apoptosis may also be treated with Glutathione. Likewise, glutathione may be useful in treating or preventing acute events which lead to pathological apoptosis of cells in the brain and elsewhere.

The preferred drug dosage form is safe, stable, is well absorbed (starting at 0.5 hours and reaching 70-80% within 1.5 hours) and readily distributed into cells.

Parkinson's Disease is a glutathione depletion disorder that occurs in the specialized brain cells in the basal ganglia, ex., the Substantia Nigra. These cells produce a powerful neurotransmitter known as dopamine. The starting substance, DOPA, and the biochemical pathway leading to dopamine production are fraught with free radical reactions that use up considerable antioxidants, including ascorbic acid and glutathione. While the initiating, etiologic agent(s) are not known, the supporting science indicates that the development and progression of the disorder involve inadequacies of glutathione. According to the present invention, glutathione levels be safely restored.

An important point for all GSH therapeutic programs is the fact that virtually all cells in the body, and especially brain cells, have active GSH transporters on their surface membranes that actively take up GSH from the surrounding tissue fluid and pump it into the cells, resulting in a major concentration increase inside the cells.

Parkinson's disease has a number of different parameters that can be quantified, and therefore clinical trials with specific End Points can be constructed and analyzed statistically for significant differences between placebo control and drug treated groups.

Glutathione is orally bioavailable, and efficiency may be increased by the administration of pharmaceutically stabilized reduced glutathione in a bolus on an empty stomach.

Glutathione is efficiently absorbed from mucous membranes, especially the sublingual mucosa and lumen of the duodenum and initial part of the ileum.

Glutathione may be administered pharmacologically, to alter a redox state within the cells of an organism, and to therefore alter an expression of redox-dependent factors, such as NM and PEDF.

Therefore, e.g., as a result of bioavailable administration of glutathione (GSH), the redox balance of the tissues will be shifted toward the reduced state. This is especially the case in the event of tissues with a high or abnormally high metabolic demand, wherein a production of free radicals is excessive. In that case, the presence of pharmacologically administered reduced glutathione will be expected to have an even greater impact in altering a redox balance in the cells. Thus, it is believed that the influence of exogenous glutathione will be particularly seen in proximity to those tissues that are at risk of ischemia.

It is noted that glutathione's effects are not limited to increasing or sustaining levels of PEDF, but rather the action of glutathione may be exerted on many different tissues and cell functions. It is particularly noted that glutathione regulated redox state may control cell function through gene induction, transcriptional, translational, posttranslational, or receptor-mediated effects, on a variety of factors.

In the case of PEDF, the administration of glutathione would be expected to act as an antineoplastic therapy by (a) reducing neovascularization, (b) serving as an influence toward differentiated states of cells, and (c) supporting the normal function of tissues, such as neurons. It is particularly noted that, in this respect, the action of glutathione as an antioxidant and free radical scavenger is believed to be distinct and separate.

In the case of NM, glutathione administration would be expected to forestall the cascade which activates certain viral replication, including HIV.

Glutathione may also alleviate certain immune and autoimmune disorders, including rheumatoid arthritis, and alter glucocorticoid effects.

It is thought that transplantation of neurons (or their precursor cells) may cure or alleviate certain pathologies. For example, in Parkinson's disease, transplantation of specific fetal brain cells into patients could alleviate or cure certain problems associated with the disease. However, the transplanted cells would have to appropriately differentiate, and remain differentiated, in situ to functionally replace the pathological or dead cells. This involves creating and maintaining a microenvironment for the cells having the appropriate growth factors and stimuli. The maintenance of a high concentration of reduced glutathione could promote, for example, the secretion of PEDF by the astroglia, or assist genetically modified (transfected) astroglia to produce high levels of PEDF, thus providing an environment rich in neural growth factors.

Ischemia Reperfusuion injury is also a particular concern in transplantation, and the pretreatment of the cells with relatively high levels of glutathione may reduce the free radical damage to the cells as well as the levels of secondary redox messengers.

As used herein, the term “pharmaceutically stabilized glutathione” refers to glutathione which is maintained in a reduced form without substantial cyclization. This stabilization may be effected by the addition of one or more agents that, together with the glutathione, provide a pharmaceutical formulation which is capable of delivering native reduced glutathione.

The present invention also includes novel combinations of glutathione and other pharmacological agents and in novel dosage forms and means for administration.

The oral administration of pharmaceutically stabilized reduced glutathione, presented as a charge transfer complex in relatively high concentration may produce a significant, predictable increase in intracellular glutathione levels in humans.

It has been found that, in otherwise healthy HIV infected humans, the intracellular glutathione levels in the peripheral blood mononucleocytes (PBMs) was significantly increased after oral administration of stabilized glutathione. This is achieved by providing a glutathione formulation which ensures delivery of adequate dose of pharmaceutically stabilized, reduced glutathione, with rapid dissolution before the duodenum. The formulation is administered to efficiently provide a high concentration of glutathione in the duodenum, i.e., on an empty stomach, to enhance uptake.

A preferred formulation includes 250 mg. or more of reduced glutathione with at least equimolar ascorbic acid, to fulfill three functions: acts as a sacrificial non-specific antioxidant during preparation, storage and after ingestion; reduces or neutralizes static electrical charge of glutathione powder, allowing dense packing of capsules; and acts as a lubricant for the encapsulation device. The ascorbic acid also maintains an acidic and reducing environment, which pharmaceutically stabilizes the glutathione molecule. Ascorbic acid is believed to form a charge couple with glutathione which enhances penetration through cell membranes, and reduces the tendency for the gamma-glutamyl and glycinyl residues to assume a cyclic conformation or to form an internal cyclic amide. The ascorbate thus complexes with the glutathione in solution to maintain a linear conformation. This linear conformation, in turn, stericly hinders the free cysteinyl thiol group. This steric hindrance stabilizes a free radical that may be formed, and thus maintains the biological activity of glutathione.

A cyclic form of glutathione, which may occur under certain conditions, such as neutral to basic pH, exposes the sulfhydryl moiety, making it more reactive. Under alkaline pH, cyclic amide formation is promoted, leaving a potentially toxic compound. The cyclic glutathione composition is a potential structural analog that may inhibit glutathione reductase, glutathione peroxidase and specific glutathione transporter proteins.

Likewise, oxidizing conditions promote disulfide formation (GSSG and Pr—S—S-G), which may reduce bioavailability of glutathione and counteract some of the potential benefits of glutathione administration. Further, oxidizing conditions also promote desulfuration, resulting in opthalmic acid formation (or other compounds), which may be toxic or inhibit efficient glutathione absorption.

A preferred oral formulation thus preferably includes an effective amount of glutathione mixed with a stabilizing agent, which is administered under such conditions that the concentration of glutathione attained in the lumen of the latter portion of the duodenum is higher than the plasma glutathione concentration, and preferably higher than the intercellular concentration of the epithelial lining cells. Thus, for example, a glutathione and ascorbic acid capsule is taken on an empty stomach. The reducing agent, preferably ascorbic acid, prevents oxidation of the glutathione during packaging and storage, and further may stabilize the glutathione in the relatively alkaline conditions of the duodenum prior to absorption. Desulfuration of glutathione leads to the formation of ophthalmic acid, the serine analog of glutathione, which inhibits glutathione uptake. This protocol is in contrast to prior art administration methods, which direct taking glutathione capsules after meals. By diluting glutathione with food, degradative enzymes are diluted and alkaline conditions buffered; however, the rapidity of absorption allows high bioavailability with only a small amount of degradation.

Glutathione may also be combined and another pharmaceutically active composition, wherein the other composition is selected from a broad group consisting of: easily oxidized compositions, antioxidant compositions, compositions with oxidant effects, compositions for the treatment of pathology associated with: suppressed total glutathione levels, suppressed reduced glutathione levels, relatively oxidized conditions in the organism, uncontrolled free radical or oxidizing reactions, or conditions where a more reduced state is desirable.

Glutathione may be used alone or in combination with other known compositions for the treatment or palliation of AIDS, HIV infection or retroviral replication (e.g., HTLV I, HTLV-II, HTLV-III, etc.), herpes virus replication (e.g., Herpes simplex type I, Herpes simplex type II, Herpes zoster (varicella), CMV, EBV, HHV-8, etc.), rabies, ebola virus, influenza virus, CHF, coronary artery disease, status post coronary artery restenosis, Diabetes mellitus, Macular Degeneration, and/or hepatitis (toxic or infectious). In addition, certain neurological conditions, such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and others may also benefit from antioxidant therapies. Further, a number of pharmaceutical therapies, especially those that cross the blood brain barrier, are associated with side effects that relate to oxidative effects. Other drugs, such as Tamoxifen, are associated with macular degeneration. Thus, glutathione may be administered to treat viral or certain bacterial infections, chronic diseases, detoxify drugs, treat or alleviate oxidative and lipid peroxidative disorders, and to reduce the long-term effects of oxidant agents, such as superoxide, which include carcinogenesis and aging.

It is noted that in the case of diseases which have as a part of their etiology a precipitation of proteins, such as amyloid diseases, e.g., Alzheimer's disease, the alteration of redox potential of the medium may have a dramatic effect on protein solubility. Thus, as the medium becomes more oxidized, the proteins will typically have more disulfide linkages, both defining the secondary structure of the peptide, and potentially forming cross linkages with other moieties. On the other hand, the administration of reduced glutathione will result in a reducing environment, with correspondingly more free sulfhydryl groups. Therefore, it is expected that administration of glutathione will provide an effective treatment, or part of a treatment regimen, for such diseases. It is also noted that precipitated peptides may be involved in free radical reactions, which will also be countered by glutathione administration.

Glutathione may also be used, alone or in combination with other therapies for the treatment of free radical associated neurological conditions, for example, Alzheimer's disease, Parkinson's disease, catecholamine toxicities, other free-radical associated toxicities, stroke and transient ischemic events, spinal chord injury and other traumatic injuries to nerve tissue, peripheral neuropathies, possibly prion-associated illness, infectious agent pathology and inflammatory pathology, or to reduce the free-radical associated toxicity of drugs administered to treat these conditions.

Mycoplasma infections, such as mycoplasma pneumonia, are believed to cause pathology due to free radical reactions within cells by these intracellular parasites. Therefore, glutathione may be administered alone or in combination with an anti-mycoplasma antibiotic for the treatment of mycoplasma infections.

Glutathione may also have benefit in the prophylaxis or treatment of pathology caused by cell-wall lacking or deficient organisms, such as mycoplasma or mycoplasma-like organisms, or L-form bacteria.

Glutathione may also be used to increase or supplement the glutathione levels in normal mammals. This may be desired, for example, for prophylaxis against ischemic events, free radical damage from sun, chemicals, or other environmental exposure, and to reduce a cancer risk.

In fact, since oxidizing conditions in an organism are generally undesirable, and where necessary the mechanisms for producing oxidizing conditions typically overpower ingested antioxidants, a large number of medications and drugs are appropriate for combination with glutathione. However, certain conditions may require care in the administration of glutathione. Further, certain cancer chemotherapy regimens rely on exhaustion of cellular free radical quenching mechanisms to selectively kill target cells. Finally, cellular apoptosis, or programmed cell death, relies on exhaustion of reduced glutathione levels in cells (mitochondria), resulting in death. Where this mechanism is required or physiologically correct, interruption by exogenous glutathione may be undesirable. Further, glutathione may interact with some compositions, either to non-specifically reduce or combine with the chemical moiety, or to alter a metabolism after ingestion; unless accounted for, these effects may be undesirable.

Glutathione may have efficacy in treatment of male infertility. Thus, glutathione may remedy mitochondrial defects or deficiency. Lenzi, A., Lombardo, F., Gandini, L., Culasso, F. & Dondero, F.: Glutathione therapy for male infertility. Arch. Androl. 29:65-68, 1992.

A known anti-HIV therapy, 3′-azidothymidine (zidovudine, AZT), acts as a potent reverse transcriptase inhibitor. This drug, however, generates free radicals and is toxic to mitochondria, and is associated with a myopathy. Glutathione may therefore be administered in conjunction with AZT to reduce toxicity while not interfering with the reverse transcriptase inhibitory activity, thus increasing the therapeutic index. Likewise, glutathione may also be used to increase the therapeutic index of other drugs that have a significant free-radical associated toxicity.

There are a number of conditions which are believed to be associated with reduced intracellular antioxidant levels, including AIDS, diabetes, macular degeneration, congestive heart failure, vascular disease and coronary artery restenosis, Herpes virus infection, toxic and infectious hepatitis, and rabies. Certain interstitial lung disease may be due to insufficient glutathione levels. Further, various toxins and medications may also result in free radical reactions, including types of cancer chemotherapy. Therefore, the administration of Glutathione holds potential to treat these diseases and conditions by the use of a convenient, effective oral formulation of glutathione. Thus, the administration of exogenous glutathione supplements the hepatic output to help maintain reduced conditions within the organism. As noted above, the failure to quench free radical reactions allows an undesirable cascade resulting in damage to macromolecules, lipid peroxidation, and generation of toxic compounds. The maintenance of adequate glutathione levels is necessary to block these free radical reactions.

Glutathione also has the ability to form complexes with metals. For example, as discussed above, glutathione forms chelation complexes with nickel, lead, cadmium, mercury, vanadium and manganese. Glutathione also forms circulating complexes with copper in the plasma. Glutathione may be administered to treat metal toxicity. It is believed that the glutathione-metal complexes will be excreted, thus reducing the metal load. Thus, glutathione may be administered for the treatment of toxicity associated with iron, copper, nickel, lead, cadmium, mercury, vanadium, manganese, cobalt, transuranic metals, such as plutonium, uranium, polonium, and the like. As compared to EDTA, glutathione has a reduced tendency to chelate calcium, providing a significant advantage. It is noted that the chelation properties of glutathione are separate from the antioxidant properties; however, some metal toxicities are free radical mediated, for example iron, and therefore glutathione administration for these conditions is particularly advantageous.

In order to provide high bioavailability, it has been found desirable to provide a relatively high concentration of reduced glutathione in proximity to the mucous membrane, e.g., the duodenum for oral administration. Thus, in contrast to prior methods, the glutathione is preferably administered as a single bolus on an empty stomach. The preferred dosage is between about 100-10,000 mg. glutathione, and more preferably between about 250-3,000 mg. glutathione. Further, the glutathione formulation is preferably stabilized with a reducing agent (antioxidant), preferably ascorbic acid, to reduce oxidation both during storage and in the digestive tract prior to absorption. The use of crystalline ascorbic acid has the added benefit of reducing the static charge of glutathione for improved encapsulation and serving as a lubricant for the encapsulation apparatus. However, other static dissipation methods or additives may be employed, and other antioxidants may be employed. The preferred dosage form is a capsule, e.g., a two-part gelatin capsule, which protects the glutathione from air and moisture, while dissolving quickly in the stomach.

The digestive tract is believed to have specific facilitated or active transport carriers for glutathione, which allow uptake of glutathione from the intestinal lumen without degradation. The uptake through this mechanism is maximized by providing a high concentration gradient and avoiding the presence or production of transport inhibitors, such as ophthalmic acid. Thus, the preferred method of oral administration employs an uptake mechanism that differs from glutathione administered using prior methods, as well as most other thiol compounds.

The oral mucosa have been found to allow rapid and efficient uptake of glutathione into the blood. In contrast to the digestive tract, the significance of facilitated or active transport mechanisms in the oral mucosa is believed to be low; rather, a high concentration of glutathione in the oral mucosa is believed to permit passive transport of the glutathione through the cells or around the cells into the capillary circulation. Therefore, compositions intended for absorption through the oral mucosa, e.g., for sublingual administration, are preferably of high purity, as contaminants may be absorbed similarly to glutathione, and as relatively small, uncharged molecules. Therefore, the composition preferably includes ascorbic acid that helps to maintain the glutathione in a reduced state, maintains a somewhat acidic environment in the mouth to avoid deprotonation of the glutamic acid residue, without causing substantially all of the amines to be protonated.

It has been found, contrary to reports of other scientists, that glutathione is not substantially degraded in the stomach, and therefore, the release of the glutathione need not diluted in the stomach or release be delayed. In fact, the glutathione formulation is preferably released and dissolved in the stomach. The addition of stabilizer, i.e., ascorbic acid, further improves the ability of the glutathione to reach its site of absorption in the intestine un-degraded. Once past the stomach, it is important that the glutathione be immediately available for absorption, as the desulfurases and peptidases from the pancreas, as well as the increase in pH, do tend to degrade the glutathione. The desulfurase produces ophthalmic acid, which interferes with glutathione absorption. Thus, by providing a high concentration of glutathione in the duodenum, without substantial dilution, the glutathione may be absorbed at a maximum rate. While the degradation of glutathione in the latter part of the duodenum and ileum may compete with the absorption process, the present method provides significant bioavailability. In fact, studies have demonstrated about 90% bioavailability of orally administered glutathione.

The capsule is preferably a standard two-part hard gelatin capsule of double-0 (00) size, which may be obtained from a number of sources. After filling, the capsules are preferably stored under nitrogen, to reduce oxidation during storage. The capsules are preferably filled according to the method of U.S. Pat. No. 5,204,114, incorporated herein by reference in its entirety, using crystalline ascorbic acid as both an antistatic agent and stabilizer. Further, each capsule preferably contains 500 mg of glutathione and 250 mg of crystalline ascorbic acid. A preferred composition includes no other excipients or fillers; however, other compatible fillers or excipients may be added. While differing amounts and ratios of glutathione and stabilizer may be used, these amounts are preferable because they fill a standard double-0 capsule, and provide an effective stabilization and high dose. Further, the addition of calcium carbonate, a component of known high dose glutathione capsules, is avoided as there may be impurities in this component. Further, calcium carbonate acts as a base, neutralizing stomach acid, which accelerates degradation of glutathione in the small intestine.

Because the glutathione and ascorbic acid are administered in relatively high doses, it is preferred that these components be highly purified, to eliminate impurities, toxins or other chemicals, which may destabilize the formulation or produce toxic effects or side effects. As stated above, the formulation may also include other pharmaceutical agents, of various classes.

Glutathione is advantageously administered over extended periods. Therefore, one set of preferred useful combinations include glutathione and drugs intended to treat chronic conditions which are well absorbed on an empty stomach, and do not have adverse interactions or reduced or variable combined absorption.

One particular class of drugs includes central or peripheral adrenergic or catecholenergic agonists, or reuptake blockers, which may produce a number of toxic effects, including neurotoxicity, cardiomyopathy and other organ damage. These drugs are used, for example, as cardiac, circulatory and pulmonary medications, anesthetics and psychotropic/antipsychotic agents. Some of these drugs also have abuse potential, as stimulants, hallucinogens, and other types of psychomimetics. Other free radical initiation associated drugs include thorazine, tricyclic antidipressants, quinolone antibiotics, benzodiazepines, acetaminphen and alcohol.

An oral pharmaceutical formulation may be made comprising glutathione in an amount of between about 50-10,000 mg, and an effective amount of a pharmacological agent capable of initiating free radical reactions in a mammal. The pharmacological agent is, for example, an adrenergic, dopaminergic, serotonergic, histaminergic, cholinergic, gabaergic, psychomimetic, quinone, quinolone, tricyclic, and/or steroid agent.

Hepatic glutathione is consumed in the metabolism, catabolism and/or excretion of a number of agents. The depletion of hepatic glutathione may result in hepatic damage or a toxic hepatitis. Such agents may include, for example, aminoglycoside antibiotics, acetominophen, morphine and other opiates. High dose niacin, used to treat hypercholesterolemia, has also been associated with a toxic hepatitis. An oral pharmaceutical formulation may be provided comprising glutathione in an amount of between about 50-10,000 mg, administered in conjunction with and an effective amount of a pharmacological agent that consumes hepatic glutathione reserves.

A number of pathological conditions result in hepatic damage. This damage, in turn, reduces the hepatic reserves of glutathione and the ability of the liver to convert oxidized glutathione to its reduced form. Other pathological conditions are associated with impaired glutathione metabolism. These conditions include both infectious and toxic hepatitis, cirrhosis, hepatic primary and metastatic carcinomas, traumatic and iatrogenic hepatic damage or resection. A pharmaceutical formulation may be provided comprising glutathione and an antiviral or antineoplastic agent. The antiviral or antineoplastic agent is, for example, a nucleoside analog.

Glutathione is broken down, and cysteine excreted, possibly in the urine. Very high doses of glutathione may therefore result in cysteinuria, which may result in cysteine stones. Other long term toxicity or adverse actions may result. Therefore, a daily intake of greater than about 10 gm. for extended period should be medically monitored. On the other hand, individual doses below about 50 mg. are insufficient to raise the concentration of the duodenal lumen to high levels to produce high levels of absorption, and to provide clinical benefit. Therefore, the preferred formulations have glutathione content greater than 50 mg, and provided in one or more doses totaling up to about 10,000 mg per day.

In the treatment of HIV infection, it is believed that the oral administration of a relatively high dose bolus of glutathione, i.e., 1-3 grams per day, on an empty stomach, will have two beneficial effects. First, HIV infection is associated with a reduction in intracellular glutathione levels in PBMs, lung, and other tissues. It is further believed that by increasing the intracellular glutathione levels, the functioning of these cells may be returned to normal. Therefore, the administration of glutathione will treat the effects of HIV infection. Therefore, glutathione and ascorbic acid may be provided in an oral formulation, optionally in combination with an antiretroviral agent. It is noted that the transcription mechanisms and control involved in retroviral infection is believed to be relatively conserved between various types. Therefore, late stage retroviral suppression is expected for the various types of human retroviruses and analogous animal retroviruses.

Second, it has been found in in vitro tests that by increasing the intracellular levels of glutathione in infected monocytes to the high end of the normal range, the production of HIV from these cells may be suppressed for about 35 days. This is believed to be related to the interference in activation of cellular transcription by cytokines, including NFκB and TNF-α. Therefore, the infectivity of HIV infected persons may be reduced, helping to prevent transmission. This reduction in viral load may also allow the continued existence of uninfected but susceptible cells in the body.

Glutathione, administered according to the present method, is believed to be effective in the treatment of congestive heart failure (CHF). In CHF, there are believed to be two defects. First, the heart muscle is weakened, causing enlargement of the heart. Second, peripheral vasospasm is believed to be present, causing increased peripheral resistance. Glutathione is effective in enhancing the effects of nitric oxide, and therefore is believed to be of benefit to these patients by decreasing vasoconstriction and peripheral vascular resistance, while increasing blood flow to the tissues. While nitroso-glutathione is more effective at preventing platelet aggregation than at vasodilation, it is nevertheless a potent vasodilator with a longer half-life than nitric oxide alone. Further, since a relative hypoxia of the tissues is associated with free radical-mediated cellular damage, the presence of glutathione will also help to block this damage. Glutathione may be orally administered in conjunction with a congestive heart failure medication, for example, digitalis glycosides, dopamine, methyldopa, phenoxybenzamine, dobutamine, terbutaline, amrinone, isoproterenol, beta blockers, calcium channel blockers, such as verapamil, propranolol, nadolol, timolol, pindolol, alprenolol, oxprenolol, sotalol, metoprolol, atenolol, acebutolol, bevantolol, tolamolol, labetalol, diltiazem, dipyridamole, bretylium, phenytoin, quinidine, clonidine, procainamide, acecainide, amiodarione, disopyramide, encainide, flecanide, lorcainide, mexiletine, tocainide, captopril, minoxodil, nifedipine, albuterol, pargyline, vasodilators, including nitroprusside, nitroglycerin, phentolamine, phenoxybenzamine, hydrazaline, prazosin, trimazosin, tolazoline, trimazosin, isosorbide dinitrate, erythrityl tetranitrate, asprin, papaverine, cyclandelate, isoxsuprine, niacin, nicotinyl alcohol, nylidrin, diuretics, including furosemide, ethacrynic acid, spironolactone, triamterine, amiloride, thiazides, bumetanide, caffeine, theophylline, nicotine, captopril, salalasin, and potassium salts.

It has been found that elevated levels of homocysteine as a significant risk in vascular disease, such as atherosclerosis, venous thrombosis, heart attack and stroke, as well as neural tube defects and neoplasia. Moghadasian et al., “Homocyst(e)ine and Coronary Artery Disease”, Arch, Int. Med. 157(10):2299-2308 (Nov. 10, 1997), incorporated herein by reference. Homocystiene promotes free radical reactions. In patients with defective homocysteine metabolism, relatively high levels of homocysteine are present in the blood. Glutathione may be administered to patients with elevated homocysteine levels.

It was believed that, because hepatocytes produce reduced glutathione through a feedback-inhibited pathway, hepatocytes would not effectively absorb reduced glutathione from the plasma. Therefore, an insult to hepatocytes, for example from toxic or infectious origin, which otherwise suppressed glutathione production, would result in cellular damage or death. In fact, the present inventors believe that this is not the case, at least in the case of compromised hepatocytes. Therefore, hepatitis, of various types, may be treated with orally administered glutathione. For example, both alcohol and acetaminophen are both hepatotoxic, and result in reduced hepatocyte glutathione levels. Therefore, these toxicities may be treated with glutathione. Glutathione may also be effective in the treatment of other types of toxicities, to various cells or organs, which result in free radical damage to cells or reduced glutathione levels. Hepatitis may also have viral etiology, and the use of glutathione may be beneficial in a similar manner to the use of glutathione in the treatment of management of HIV infection. The glutathione may act to reduce expression of viral genes, as well as reduce the oxidative challenge resulting from active viral replication. Glutathione may also reduce viral disulfide bonds, reducing viral infectivity.

Diabetes, especially uncontrolled diabetes, results in glycosylation of various enzymes and proteins, which may impair their function or control. In particular, the enzymes which produce reduced glutathione (e.g., glutathione reductase) become glycosylated and non-functional. Therefore, diabetes is associated with reduced glutathione levels, and in fact, many of the secondary symptoms of diabetes may be attributed to glutathione metabolism defects. Glutathione may therefore be applied to supplement diabetic patients in order to prevent the major secondary pathology. An oral pharmaceutical formulation comprising glutathione and an anti-hyperglycemic agent may also be administered to a patient in need of such treatment.

Glutathione, due to its strong reducing potential, breaks disulfide bonds. It is believed that most normal proteins are not denatured, to a great extent, by normal or superphysiologic levels of glutathione. It is believed, however, that opiate receptors are deactivated by high normal levels of glutathione. It is therefore believed that glutathione administration may be of benefit for the treatment of obesity and/or eating disorders, other addictive or compulsive disorders, including tobacco (nicotine) and opiate additions.

Glutathione may be administered in conjunction with nicotine. The physiologic effects of nicotine are well known. Glutathione, due to its vasodilatory effects, improves cerebral blood flow, resulting in a synergistic cerebral function-enhancing effect.

In mammals, the levels of glutathione in the plasma are relatively low, in the micromolar range, while intracellular levels are typically in the millimolar range. Therefore, the intracellular cytosol proteins are subjected to vastly higher concentrations of glutathione than extracellular proteins. The endoplasmic reticulum, a cellular organelle, is involved in processing proteins for export from the cell. It has been found that the endoplasmic reticulum forms a separate cellular compartment from the cytosol, having a relatively oxidized state as compared to the cytosol, and thereby promoting the formation of disulfide links in proteins, which are often necessary for normal activity. Hwang, C., et al., “Oxidized Redox State of Glutathione in the Endoplasmic Reticulum”, Science 257:1496-1502 (11 Sep. 1992), incorporated herein be reference. In a number of pathological states, cells may be induced to produce proteins for export from the cells, and the progression of the pathology interrupted by interference with the production and export of these proteins. For example, many viral infections rely on cellular production of viral proteins for infectivity. Interruption of the production of these proteins will interfere with infectivity. Likewise, certain conditions involve specific cell-surface receptors, which must be present and functional. In both these cases, cells that are induced to produce these proteins will deplete reduced glutathione in the endoplasmic reticulum. It is noted that cells that consume glutathione (GSH) will tend to absorb glutathione from the plasma, and may be limited by the amounts present. Therefore, by increasing plasma glutathione levels, even transiently, the reducing conditions in the endoplasmic reticulum may be interfered with, and the protein production blocked. Normal cells may also be subjected to some interference; however, in viral infected cells, or cells abnormally stimulated, the normal regulatory mechanisms may not be intact, and the redox conditions in the endoplasmic reticulum controlled by the availability of extracellular glutathione. In these conditions, the pharmaceutical administration of glutathione may produce significant effects.

Reproduction of herpes viruses, which are DNA viruses, is inhibited or reduced in cell culture by the administration of extracellular glutathione. Herpes virus infections may be treated by administering glutathione. The known herpes viruses include herpes simplex virus I, herpes simplex virus II, herpes zoster, cytomegalovirus, Epstein Barr virus, as well as a number of other known viruses.

It is also believed that infection by the rabies virus, an RNA virus, may be treated by the administration of glutathione. While standard treatments are available, and indeed effective when timely administered, glutathione may be useful in certain circumstances. Therefore, rabies virus infection may be treated, at least in part. One available treatment for rabies is an immune serum. Glutathione may be parenterally administered in combination with an antibody, such as a monoclonal antibody, humanized antibody, or donor antibodies derived from human or animal sources. Glutathione may also be administered separately.

Coronary heart disease risk is increased by the consumption of a high-fat diet, and reduced by the intake of antioxidant vitamins, including vitamin E and vitamin C, as well as flavonoids. High fat meals impair the endothelial function through oxidative stress, resulting in impaired nitric oxide availability. It has been found that vitamin C and vitamin E restores the vasoconstriction resulting from nitric oxide production by endothelium after a high fat meal. Plotnick, G. D. et al., “Effect of Antioxidant Vitamins on the Transient Impairment of Endothelium-Dependent Brachial Artery Vasoactivity Following a Single High Fat Meal”, JAMA 278:1682-1686 (Nov. 26, 1997), incorporated herein by reference. Glutathione may be administered as a prophylaxis against vascular disease.

In utilizing antioxidants as advanced therapeutic approaches, the following principles have been developed over time: Different disorders generate different types of free radicals, in different environments. Therefore, different specific antioxidants are needed for these various radicals and related compounds. The commonest species and related molecules includes superoxide, .O₂—; hydroxyl, .OH; peroxy, .OOH; hydrogen peroxide, H₂O₂ (splitting into hydroxyl radicals); alkoxy, RO.; delta singlet oxygen, ¹O₂; nitric oxide, .NO; lipid hydroperoxides, LOOH (splitting into alkoxy and hydroxyl radicals). See, Montagnier, Luc, Olivier, Rene, Pasquier, Catherine (Eds.), Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases, Marcel Dekker, NY (1998), incorporated herein by reference in its entirety.

In addition to qualitative differences among several species of free radicals, their rates of formation will differ, as will the different types of inciting agents that may have to be simultaneously controlled. For example, continued, unprotected exposures of the eyes, in Macular Degeneration, to strong sunlight and to tobacco smoke, would limit benefits from an antioxidant used as a therapeutic agent for control of this disease. Synergistic therapies may be provided to patients by increasing antioxidant levels systemically or in specific organs as well as reducing oxidative, free radical generating and ionizing influences. In this case, glutathione therapy would be complemented with ultraviolet blocking sunglasses, and a tobacco smoking cessation plan, if necessary. A particularly advantageous antioxidant for combination with glutathione is alpha tocopherol succinate.

Free radicals occur in different parts or subparts of tissues and cells, with different inciting agents. For example, in trauma to the brain or spinal cord, the injurious free radicals are in the fatty (lipid) coverings that insulate nerve fibers, the myelin sheaths. Extremely high doses of a synthetic corticosteroid, 5 to 10 grams of methyl prednisolone sodium succinate (MPSS), given for just 24 hours, rapidly reach the brain and spinal cord and diffuse rapidly into the myelin, neutralizing the trauma-induced radicals, specifically: .OH, .OOH, and RO. A pharmaceutical composition may be provided comprising a combination of glutathione and a glucocorticoid agent.

TRX has been shown to modulate the signaling processes of programmed cell death (apoptosis). TRX and other thiol compounds exert a protective activity against cytotoxicity and apoptosis induced by various oxidative stresses. For example, Fas and TNF-α dependent cell death may be protected by intracellular as well as extracellular TRX. The activity of the ICE (interleukin 1 b-converting enzyme) family proteases (caspases), with cysteine residue in their active site, which are involved in apoptosis, are regulated by a redox mechanism. For example, the activity of caspase-3 (CPP32), an important member of caspases, is markedly inhibited by oxidizing agents, which is counteracted by dithiothreitol or TRX. In contrast, on exposure to diamide or hydrogen peroxide, a marked increase of CPP32 protease activity was observed after a few hours, suggesting that intracellular redox state profoundly modulates the signaling processes of apoptosis by regulating the activity of caspases. Many transcription factors and DNA-binding proteins are redox regulated by TRX, including NM, AP-1, PEBP2/AML-1, and p53. Junji Yodoil, Shugo Ueda, Masaya Ueno, Tetsuro Sasada, and Hiroshi Masutani, Redox control of Thioredoxin (TRX) on the cytotoxic/death signal.

Superoxide (O₂ ⁻) is the compound obtained when oxygen is reduced by one electron. Oxidants related to superoxide include H₂O₂ and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These molecules are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Oxidizing agents are, however, are physiologically important for host defense, where they serve as microbicidal and parasiticidal agents, in normal apoptosis, or programmed cell death, and in biological signaling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins. It is generally believed that host defense mechanisms are mediated by such strong effects that pharmacological antioxidants would not be able to overcome the powerful oxidant effects. On the other hand, it is believed that antioxidant pharmaceuticals may play an important role in modulating redox-mediated signaling and early steps in biological cascades, such as apoptosis.

The accepted, published, peer-reviewed literature has repeatedly demonstrated the multiple properties of glutathione in the body. The abundant physiological and biochemical properties of glutathione led others into an extensive series of clinical trials wherein precursors of glutathione were administered, because the prevailing belief was that glutathione itself could not be effectively absorbed if it was simply given as glutathione. Hence, the popularity of the relatively ineffective and potentially damaging glutathione precursor N-acetyl cysteine (NAC) is currently being misused in the homosexual (high AIDS risk) community. The further belief was that glutathione would not cross the membranes of lymphocytes and other cells, whereas NAC could. The view was that to try to correct the glutathione deficiency in HIV/AIDS, with glutathione itself, was a hopeless task, because it would be degraded before uptake across membranes. However, the precursors of glutathione have failed to raise intracellular GSH levels. A suitable regimen for oral administration of glutathione to achieve high bioavailability and increased intracellular levels of glutathione is provided.

While prior studies have employed glutathione dissolved in orange juice to administer glutathione to AIDS patients, resulting in glutathione uptake, this method does not provide the advantages of an encapsulated or pill form, and there was no recognition for the need to prevent digestive dilution or glutathione derived impurities from being present.

Glutathione has also proven to be an effective anti-viral agent and interferes with HIV replication at a critical site that is not affected by other current drugs, viral mRNA transcription. Glutathione keeps viral DNA quiescent, especially when potent activators are present, like NFκB, and TNF-α. Glutathione's anti-viral target appears to be at a point where the virus can not readily mutate. The dependence of HIV replication on binding activated NFκB onto its Long Terminal Repeat (LTR) appears to be central to the virus.

Orally administered glutathione can safely raise cell levels beyond correcting glutathione deficiencies. A number of pathologic processes can be inhibited by these higher levels, for example, curtailing the virtually self-perpetuating, powerful biochemical cycles producing corrosive free radicals and toxic cytokines that are largely responsible for the signs and symptoms of AIDS. These biochemical cycles destroy considerable quantities of glutathione but they can eventually be brought under control, and normalized with sufficient, on-going glutathione therapy. A typical example is the over production of a substance, 15 HPETE (15-hydroperoxy eicosatetraenoic acid), from activated macrophages. The 15 HPETE is a destructive, immunosuppressing substance and requires glutathione for conversion into a non-destructive, benign molecule. The problem is that once macrophages are activated, they're difficult to normalize.

Once inside cells, GSH curtails the production of free radicals and cytokines, corrects the dysfunctions of lymphocytes and of macrophages, reinforces defender cells in the lungs and other organs, halts HIV replication in all major infected cell types, by preventing the activation of the viral DNA by precluding the activation of NM, inhibits the TAT gene product of HIV that drives viral replication, dismantles the gp120 proteins of the virus coat. These gp120 proteins are the projections of the virus that normally allow it to lock onto susceptible CD4+ cells thereby helping the spread of the virus to uninfected CD4+ cells. By disrupting the gp120 protein, glutathione offers a potential mode of preventing transmission not only to other cells in the patient, but perhaps in precluding transmission to others.

Besides classic antiviral or antiretroviral agents (reverse transcriptase inhibitors, protease inhibitors), a number of other therapies may be of benefit for AIDS patients, for example combinations of glutathione with the following drugs: cyclosporin A, thalidomide, pentoxifylline, selenium, desferroxamine, 2L-oxothiazolidine, 2L-oxothiazolidine-4-carboxylate, diethyldithiocarbamate (DDTC), BHA, nordihydroguairetic acid (NDGA), glucarate, EDTA, R-PIA, α-lipoic acid, quercetin, tannic acid, 2′-hydroxychalcone, 2-hydroxychalcone, flavones, α-angelicalactone, fraxetin, curcurmin, probucol, and arcanut (areca catechul).

Inflammatory responses are accompanied by large oxidative bursts, resulting in large numbers of free radicals. Therefore, glutathione may have application in the therapy for inflammatory diseases. Glutathione may advantageously reduce the primary insult a well as undesired aspects of the secondary response. Glutathione may be administered to patients suffering from an inflammatory disease process, such as arthritis or various types, inflammatory bowel disease, etc. Combination pharmaceutical therapy may be provided including glutathione and an analgesic or antiinflammatory agent, for example opiate agonists, glucocorticoids or non-steroidal antiinflammatory drugs (NSAIDS), including opium narcotics, meperidine, propoxyphene, nalbuphine, pentazocine, buprenorphine, asprin, indomethacin, diflunisal, acetominophen, ibuprofen, naproxen, fenoprofen, piroxicam, sulindac, tolmetin, meclofenamate, zomepirac, penicillamine, phenylbutazone, oxyphenbutazone, chloroquine, hydroxychloroquine, azathiaprine, cyclophosphamide, levamisole, prednisone, prednisolone, betamethasone, triamcinolone, and methylprednisolone.

Glutathione may also hold benefit for the treatment of parotitis, cervical dysplasia, Alzheimer's disease, Parkinson's disease, aminoquinoline toxicity, gentamycin toxicity, puromycin toxicity, aminoglycoside nephrotoxicity, paracetamol, acetaminophen and phenacetin toxicity.

Glutathione need not be orally ingested in order to provide the beneficial effects noted. While the drug may be administered intravenously or parenterally, it may also be administered through mucous membranes, including sublingually, as a vaginal or rectal suppository, and by pulmonary inhaler, for topical applications to the alveolar surface cells of the lungs to enhance pulmonary protection against unusual pneumonias. Systemic administration of glutathione may be used to concentrate glutathione in lymph nodes, and lymphoid tissues.

Glutathione tends to be unstable in solution. Therefore, a pharmaceutical administration apparatus is provided having a dual chamber distribution pouch, having a frangible interconnection, allowing mixing between an aqueous phase and a dry glutathione preparation. The aqueous phase may be, for example, a gel, cream or foam. Either pouch may also contain another pharmaceutical agent, as described above.

A glutathione administration appliance may be used for delivering an effective dose of glutathione to an accessible mucous membrane, such as the oral, vaginal, urethral or anal cavities. A dry glutathione preparation, for example in a dehydrated gel, matrix or polymer, having a high surface area per unit volume ratio, is provided in a foil bag or pouch. The dehydrated mass includes glutathione, as well as an optional stabilizing agent, such as ascorbic acid. The dehydrated mass is hydrated by the mucosal membrane or by an externally applied fluid, and the glutathione is then present to protect the mucous membrane from viral infection.

The ability of glutathione to chemically dismantle the gp120 protein of HIV by chemically destroying structural disulfide bonds, indicates that transmission of the infection may be curtailed to some extent. If gp120 is dismantled, the virus cannot lock onto CD4+ cells. The oral glutathione treatment of patients may suffice to dismantle gp120 of viruses from treated patients. The topical applications of glutathione to mucous membranes might possibly serve to protect a sex partner if unsafe sexual practices occur.

Another effect is seen when glutathione or nitroso-glutathione is placed in the male urethra. In this case, the glutathione or glutathione derivative is absorbed. The vasodilatory effects of nitroso-glutathione, which is formed by interaction of glutathione with nitric oxide or provided directly, vasodilates the penis, resulting in an erection. Thus, a urethral glutathione or nitroso-glutathione suppository has potential for the treatment of impotence. Glutathione or nitroso-glutathione may also be used to treat female sexual dysfunction. Direct application of glutathione or nitroso-glutathione to the mucous membranes, for example, as a cream or in a gel formulation, will result in local vasodilation, lubrication, and engorgement of erectile tissue.

It is noted that the effects of various pharmacological agents which act to increase the production of nitric oxide, for example the substrate for formation of nitric oxide, the amino acid arginine, the stability of nitric oxide in the blood, or the effect of nitric oxide, may be used synergistically. Likewise, drugs which act on differing systems, such as the central nervous system and peripheral vascular system, may also be used synergistically. Thus, glutathione may be used alone or in combination to achieve its effects on the circulatory system and vascular tissues.

Glutathione or a glutathione derivative may also be co-administered with yohimbine, an alpha-2 receptor blocker, providing a synergistic effect. Yohimbine has been established to treat male sexual dysfunction, (e.g., impotence), among other effects. Apomorphine may also provide synergistic effects with glutathione for the treatment of impotence. It is noted that, in many cases, female sexual dysfunction may be related to pelvic and genital vascular response, in particular vasodilation, and therefore glutathione, alone or in combination with other vasoactive or neuroactive substances, may be beneficial in the treatment of both male and female sexual dysfunction.

Glutathione may be administered to mucous membranes in the form of a liquid, gel, cream, jelly, absorbed into a pad or sponge. Administration may also be provided by a powder or suspension.

The effective delivery of intact, pharmaceutically stabilized, bioavailable reduced L-glutathione has been accomplished. By providing high-dose glutathione for the body's general use, diabetics having either form of the disease may be provided with ample supplies of glutathione. Correcting the glutathione deficiency and also raising the levels inside cells to the upper range of normal will help to delay, or prevent the complications of diabetes.

Glutathione, orally administered, in moderately high doses, one to five gm/day, may be able to affect the outcome of macular degeneration. The avidity with which the RPE cells take up glutathione indicates that they may have a critical role in ameliorating this disorder. Unlike rods and cones, RPE cells can divide and replenish themselves if allowed. If caught at an early stage, before significant losses of rods and cones, the condition may be halted and delayed possibly indefinitely.

Since glutathione is relatively non-toxic, it may be used liberally for its advantageous properties. Glutathione may be added to a viral contaminated fluid or potentially contaminated fluid to inactivate the virus. This occurs, for example, by reduction of critical viral proteins. According to a preferred embodiment, glutathione is added to blood or blood components prior to transfusion. The added glutathione is in the reduced form, and is added in a concentration of between about 100 micromolar to about 500 millimolar or to a solubility limit, whichever is lower, and more preferably in a concentration of about 10-50 millimolar.

The addition of glutathione to whole blood, packed red blood cells or other formed blood components (white blood cells, platelets) may be used to increase the shelf life and/or quality of the cells or formed components.

It is also noted that other pharmacological agents may be employed to achieve alterations in redox balance or to acts free radical scavenging agents. These may be employed individually or in combination. For example, glutathione may also be administered in conjunction with other antioxidants or redox-active drugs; a preferred formulation for oral administration of glutathione includes ascorbic acid (Vitamin C). Other acceptable agents for administration include α-tocopherol, either in the free state as an antioxidant or as a pharmaceutically acceptable ester thereof as a Vitamin E precursor. In addition, α-

lipoic acid is believed to be a nontoxic, orally bioavailable and effective antioxidant. It is noted, however, that glutathione is a most preferred agent due to its central role in maintaining cell oxidative balance, ubiquity in the body, and high therapeutic index. One traditional difficulty, obtaining high oral bioavailability for glutathione, has been solved.

Summary of Phase 1 and Early Phase 2 HIV Results

The inventor has developed and has successfully clinically tested a safe, stabilized, orally bioavailable formulation of glutathione (L-gamma glutamylcysteinyl glycine, reduced). This achievement had not been thought to be possible because: a high density of molecular charges precluded trans membrane transport (12); the thiol group is at some risk, and if there is extended oxidation, desulfuration may occur, with the consequent formation of ophthalmic acid, a toxic structural analogue that blocks mitochondrial glutathione transporters (115); a commonly believed view held that the two amino peptide bonds would be hydrolyzed immediately by gastric acid, and/or the gastric and pancreatic proteases.

The preferred formulation and protocol for administration of Glutathione: dissipates the high density of molecular charges, without altering the structure or chemical properties of glutathione, using basic concepts in chemical physics that resulted in the issuance of U.S. and international patents; the thiol group was chemically stabilized, with no evidence of oxidative desulfuration during manufacture, storage, or in patient use at prolonged, high doses of 0 to 75 mg/kg, per day, divided into two equally divided doses; gastric acid in the human stomach is incapable of hydrolyzing the amino peptide bonds because such hydrolysis requires 6N HCl, and a temperature of 110° C. for 18 hours; the glutathione formulation withstood acid degradation for 23 hours at pH 1.2; the proteases encountered by glutathione in the stomach and in the duodenum do not attack these two amino peptide bonds; this type of information has served initial amino acid sequence efforts of proteins in the past: for example, trypsin hydrolyzes the carboxyl side of arginine and lysine residues; chymotrypsin is selective for the carboxyl side of the aromatic chains of tyrosine, tryptophane and phenylalanine; carboxypeptidase A hydrolyzes carboxyl terminals if the residue has an aromatic or bulky, aliphatic side chain; glutathione is absorbed, intact, as a tripeptide over a short segment of upper jejunum, starting approximately at the Ligament of Treitze; however, further down in the small intestine, the non specific endopeptidases do hydrolyze glutathione into its three components. The dosing rationale of high doses of “charge-neutered” glutathione between meals, is effective as shown in the Areas Under the Curve (AUC's), below.

Early clinical trials determined the pharmacokinetics of absorption and intracellular distribution in Peripheral Blood Mononuclear Cells in asymptomatic HIV (+) patients, using three doses of 1 gm, 2 gms and 3 gms/day over a 4 week period, with two weeks of baselines, plus two weeks of post drug exposure. Approximately 400 glutathione assays were carried out on each patient, including 24-hour urine samples.

A total of 10,000 glutathione assays were performed. The independent analyses demonstrated a dose response relationship. The glutathione was absorbed and distributed within the PBMC, starting at

30 minutes after ingestion, and completed in 60 minutes. This study demonstrated safety, and good patient acceptance, with no evidence of toxicities.

Examples of Areas Under the Curve (AUC) are appended, together with the results of a separate small clinical study on far advanced AIDS patients who had exhausted the HAART combinations and averaged 300,000 copies (HIV RNA PCR) and 50 CD4+ cells/per cubic mm. The latter small group consumed 75 mg/kg in two equally divided doses for 3.0 years. Survival for the four who followed the regimen extended for 3 years after starting on GSH. Two individuals who started the regimen at approximately the same time as the four illustrated on the following pages, but who dropped out, succumbed within 6 months. The four patients illustrated did not restore their CD4+ cell counts to any extent. GSH compliance began to fail between the second and third years.

Absorption Glutathione (GSH) into Cells

The Phase I/II data, was based on approximately 10,000 glutathione assays of peripheral blood mononuclear cells (PBMC's) of HIV positive people given the drug regimen. A dose response relationship was demonstrated, and correction of the intracellular glutathione insufficiency was evident.

Example 1

Reduced L-glutathione, a naturally-occurring water-soluble tripeptide (gamma-glutamyl-cysteinyl-glycine) is the most prevalent intracellular thiol in most biological systems. A preferred formulation of glutathione according to the present invention provides capsules for oral use containing 500 mg reduced L-glutathione, 250 mg USP grade crystalline ascorbic acid, and not more than 0.9 mg magnesium stearate, NF grade in an 00-type gelatin capsule. The powder may also be packaged in packets, for example containing 500 mg to 5 gm, and more preferably 1-3 grams per packet. The preferred packet preferably forms an oxygen impermeable barrier, to maintain the glutathione in a substantially reduced state for at least about 2.5 years under standard temperature and pressure conditions. For example, a metallized (e.g., aluminized), heat sealable polymer film packet may be suitable.

Example 2

The preferred regimen for treatment of humans with glutathione according to the present invention is the administration of between 1 and 10 grams per day, in two divided doses, between meals (on an empty stomach), of encapsulated, stabilized glutathione according to Example 1. The study detailed in Appendix B administered the glutathione to HIV infected, otherwise healthy males between 18 and 65, with CD4+ cell counts above 500, not on any other medications. Clinical responses were seen in the PBM intracellular glutathione levels. Thus, at 1 hour after administration of a 1-gram bolus of encapsulated stabilized glutathione in two 500 mg capsules, a three-fold increase in glutathione was measured. It is noted that, since the human body produces large quantities of glutathione, the effects of external glutathione in individual cases may sometimes be masked or even appear paradoxical. However, a statistical analysis shows a dose response effect of the administration of glutathione to the subject population.

Example 3

24 HIV positive people received glutathione in a clinical trial. The first dose of one gram was taken at 0 time, or 10:00 a.m., and the second dose at 3 hours, or 1:00p.m. A baseline was measured two weeks earlier, on the same patient. A temporary intravenous catheter was in place for 7 hours to permit frequent blood sampling at the numerous time points. The statistical analysis of the entire patient population shows statistically significant elevations and a significant dose response relationship. In a compressed Phase I/II clinical trial (FDA IND#45012), in a well-defined GSH deficiency state, HIV infection, the composition according to Example 1 administered according to the protocol of Example 2 was demonstrated to rapidly and safely raises intracellular GSH levels two to three fold. Thus, by employing the composition according to Example 1 administered according to the protocol of Example 2, an oral pharmaceutical has been shown to treat the critical losses of GSH that are known to propel a range of major disorders. The glutathione metabolism, especially the pharmacokinetics, of the subjects of the Phase II study is believed to be relatively normal. Therefore, the same regimen may be applied in the treatment of other conditions, including CHF, diabetes, early stroke or other ischemic event, toxic insult, viral infection or disease, or other condition in which free radical reactions are uncontrolled, aberrant, or contribute to pathology.

Example 4 Combination of Glutathione and Acetaminophen

A combination pharmaceutical is provided to ameliorate the detrimental effects of acetaminophen, a drug which consumes glutathione in the liver during metabolism, and in excess doses causes liver damage due to oxidative damage. The composition includes 500 mg L-glutathione, 250 mg crystalline ascorbic acid, and 350 mg acetaminophen.

Example 5 Combination of Glutathione and Chlorpromazine

A combination pharmaceutical is provided to ameliorate the detrimental effects of chlorpromazine, a phenothiazine drug that causes side effects, including tardive dyskinesia, possibly relating to excess free radical reactions. The composition includes 500 mg L-glutathione, 250 mg crystalline ascorbic acid, and 200 mg chlorpromazine.

Example 6 Combination of Glutathione and Aminoglycosides

A combination pharmaceutical is provided to ameliorate the detrimental effects of Aminoglycoside drugs, which include, but are not limited to, neomycin, kanamycin, amikacin, streptomycin, gentamycin, sisomicin, netilmicin and tobramycin, a drug class which may be associated with various toxicities. This damage may be related to oxidative damage or consumption of glutathione during metabolism. The composition is an intravenous formulation, including the aminoglycoside in an effective amount, and L-glutathione in an amount of about 10-20 mg/kg. Ascorbic acid in an amount of 5-10 mg/kg may be added as a stabilizer.

Example 7 Urethral Insert

A composition containing 200 mg glutathione, 50 mg ascorbic acid per unit dosage is mixed with carageenan and/or agarose and water in a quick-gelling composition, and permitted to gel in a cylindrical form having a diameter of about 3 mm and a length of about 30 mm. The composition is then subjected to nitric oxide to cause between 0.1-10% of the glutathione to be converted to nitroso-glutathione. The gelled agarose is then freeze-dried under conditions which allow shrinkage. The freeze-dried gel is than packaged in a gas barrier package, such as a foil pouch or foil “bubble-pack”. The freeze-dried gel may then be used as a source of nitroso-glutathione for administration transmucosally. The cylindrical freeze-dried gel may be inserted into the male urethra for treatment of impotence, or administered sublingually for systemic vasodilation.

Example 8 Vascular Disease Prophylaxis

An oral formulation is provided for prophylaxis of vascular disease, e.g., in men over 40. The composition includes 500 mg reduced L-glutathione, 250 mg USP grade crystalline ascorbic acid, and 50 mg USP acetyl salicylic acid (aspirin) in an 00-type gelatin capsule. Typical administration is twice per day. Advantageously, the acetyl salicylic acid may provided in enteric release pellets within the capsule, slowing release.

Example 9 Vascular Disease Prophylaxis

Arginine is the normal starting substrate for the production of nitric oxide. Arginine is normally in limited supply, and thus a relative deficiency of arginine may result in impaired vascular endothelial function. An oral formulation is provided for prophylaxis of vascular disease. The composition includes 500 mg reduced L-glutathione, 200 mg USP grade crystalline ascorbic acid, and 200 mg arginine, in an 00-type gelatin capsule.

Example 10 Vascular Disease Prophylaxis

Vitamin E consumption reduces the risk of heart attack and other vascular disease. Vitamin E succinate (α-tocopherol succinate) is a dry powder. An oral formulation is provided for prophylaxis of vascular disease. The composition includes 500 mg reduced L-glutathione, 200 mg USP grade crystalline ascorbic acid, and 200 mg vitamin E succinate, in an 00-type gelatin capsule.

Example 11 Vascular Disease Prophylaxis

Nonspecific esterases are present in the plasma that have a broad substrate specificity. Esters are formed between agents that are useful combination therapies, in order to provide for efficient administration, high bioavailability, and pharmaceutical stability. Preferred esters include α tocopherol-ascorbate, a tocopherol-salicylate, and ascorbyl-salicylate. The tocopherol ester maintains the molecule in a reduced state, allowing full antioxidant potential after ester cleavage. These esters may be administered alone or in combination with other agents, for example glutathione. Typically, these are administered to deliver an effective dose of salicylate equivalent of 100 mg per day for prophylaxis or 750-1000 mg per dose for treatment of inflammatory diseases. Tocopherol is administered in an amount of 100-500 IU equivalent. Ascorbate is administered in an amount of up to 1000 mg equivalent. In order to enhance availability, a non-specific esterase may be provided in the formulation to cleave the ester after dissolution of the capsule. Therefore, a non-specific esterase, such as a bacterial or Saccharomyces (yeast) enzyme or enriched enzyme preparation may be included in the formulation, such as included as a powder or as pellets in the capsule.

Example 12 Vascular Disease Prophylaxis

Nordihydroguaretic acid is a known lipoxygenase inhibitor. This composition may therefore be used to treat inflammatory processes or as prophylaxis against vascular disease. An oral formulation is provided for prophylaxis of vascular disease. The composition includes 500 mg reduced L-glutathione, 200 mg USP grade crystalline ascorbic acid, and 100 mg nordihydroguaretic acid, in an 00-type gelatin capsule. Typical administration is twice per day. The references and patents hereinabove recited are expressly incorporated herein by reference.

Example 13 Prophylaxis

Glutathione packets containing mixed 2,500 mg reduced L-glutathione, and 750 mg USP grade ascorbic acid powder are administered twice per day, by mouth, to otherwise healthy adult humans. The powder may be absorbed sublingually, swallowed as a bolus on an empty stomach, or dissolved in a liquid, such as water or orange juice, and drunk, on an empty stomach.

Example 14 Prophylaxis

Glutathione packets containing mixed 1,000 mg reduced L-glutathione, and 500 mg USP grade ascorbic acid powder are administered, 1-2 packets, twice per day, by mouth, to otherwise healthy human children. The powder may be absorbed sublingually, swallowed as a bolus on an empty stomach, or dissolved in a liquid, such as water or orange juice, and drunk, on an empty stomach.

Example 15 Treatment

Glutathione is administered orally on an empty stomach, if tolerated, or intravenously, to affected individuals suffering from acute exposure to radiation or radioactive materials, biowarfare agents, chemical warfare agents, or sepsis. Dose is 5-20 grams per day, in divided doses or as a periodic or continuous infusion.

Example 15 Treatment

Glutathione is administered orally on an empty stomach to affected patients suffering from chronic neurological disease, including but not limited to Alzheimer's disease or Parkinson's disease. Dose is 2-10 grams per day, in two divided doses.

It should be understood that the preferred embodiments and examples described herein are for illustrative purposes only and are not to be construed as limiting the scope of the present invention, which is properly delineated only in the appended claims. 

What is claimed is:
 1. A method of treating or propylaxing a mammal against at least one of nuclear, biological and chemical threats, comprising orally administering a pharmaceutically acceptable formulation of glutathione, or a glutathione salt or derivative, in sufficient amount to prophylax against or treat such threats.
 2. The method according to claim 1, wherein the threat comprises at least two of nuclear, biological and chemical.
 3. The method according to claim 1, wherein the threat comprises each of nuclear, biological and chemical.
 4. The method according to claim 1, wherein the threat comprises a radioactive element threat which damages tissues through ionizing radiation.
 5. The method according to claim 1, wherein the threat comprises a chemical threat which damages tissues through generation of free radical reactions.
 6. The method according to claim 1, wherein the threat comprises a chemical threat which comprises a chemical warfare agent.
 7. The method according to claim 1, wherein the threat comprises a mustard gas chemical threat.
 8. The method according to claim 1, wherein the threat comprises a biological threat whose pathology is associated with an oxidative stress on the host organism.
 9. The method according to claim 1, wherein the threat comprises a biological threat whose replication is promoted by oxidized cellular conditions.
 10. The method according to claim 1, wherein the threat comprises a biological threat which comprises a bioterror agent.
 11. The method according to claim 1, wherein the threat comprises a smallpox biological threat.
 12. The method according to claim 1, wherein the threat comprises an anthrax biological threat.
 13. The method according to claim 1, wherein the threat comprises a biological threat which is associated with sepsis.
 14. A pharmaceutical formulation for treatment or prophylaxis of nuclear, biological and chemical threats, comprising an orally bioavailable formulation of glutathione or a pharmaceutically acceptable salt or derivative thereof, provided in sufficient quantity to intercede to prevent death of a human as a result of said threats.
 15. The pharmaceutical formulation according to claim 14, wherein said glutathione is provided in an amount of at least one gram per unit dose.
 16. The pharmaceutical formulation according to claim 14, wherein said formulation is provided as a bulk powder stabilized against oxidation by a sacrificial antioxidant. 